Method to produce a highly concentrated immunoglobulin preparation for subcutaneous use

ABSTRACT

The present invention relates to a new and improved method for preparing a highly concentrated immunoglobulin composition from pooled plasma for subcutaneous injection. A composition comprising 20% or more immunoglobulin suitable for subcutaneous use is also described.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/789,345, filed May 27, 2010, which claims the benefit of U.S.Provisional Application No. 61/181,606 filed May 27, 2009, which areexpressly incorporated herein by reference in their entirety for allpurposes.

BACKGROUND OF THE INVENTION

Immune globulin products from human plasma were first used in 1952 totreat immune deficiency. Initially, intramuscular or subcutaneousadministration of IgG were the methods of choice. For injecting largeramounts of IgG necessary for effective treatment of various diseases,however, the intravenous administrable products with lower concentratedIgG (50 mg/mL) were developed. Usually intravenous immunoglobulin(IVIG), contains the pooled immunoglobulin G (IgG) immunoglobulins fromthe plasma of more than a thousand blood donors. Typically containingmore than 95% unmodified IgG, which has intact Fc-dependent effectorfunctions, and only trace amounts of immunoglobulin A (IgA) orimmunoglobulin M (IgM), IVIGs are sterile, purified IgG productsprimarily used in treating three main categories of medicalconditions: 1. immune deficiencies such as X-linked agammaglobulinemia,hypogammaglobulinemia (primary immune deficiencies), and acquiredcompromised immunity conditions (secondary immune deficiencies),featuring low antibody levels; 2. inflammatory and autoimmune diseases;and 3. acute infections.

A number of IVIG commercial suppliers provide a variety of IVIGproducts. Compared to the older lyophilized IVIG products containingonly 50 mg/mL protein in the solution after re-dissolving, the latestdevelopments are 100 mg/mL ready-for-use sterile, liquid preparation ofhighly purified and concentrated human IgG antibodies. Since IgGproducts such as IVIGs are manufactured from pooled human plasma,pathogen contamination (especially viruses known to cause variousdiseases in human) from donor blood is a serious concern in theproduction process. Another important consideration in IgG products istheir stability during storage, especially as ready-for-usepreparations. Compared to IVIG, subcutaneously administrableimmunoglobulin preparations have the advantages of home-care treatmentpossibility and less side effects. To reduce the disadvantage of thesmall injection volume per site, a higher concentrated IgG (e.g.,containing 200 mg/mL instead of 100 mg/mL) would be a clear advantage.

In the fourth installment of a series of seminal papers published on thepreparation and properties of serum and plasma proteins, Cohn et al. (J.Am. Chem. Soc., 1946, 68(3): 459-475) first described a methods for thealcohol fractionation of plasma proteins (method 6), which allows forthe isolation of a fraction enriched in IgG from human plasma. Severalyears later, Oncley et al. (J. Am. Chem. Soc., 1949, 71(2): 541-550)expanded upon the Cohn methods by publishing a method (method 9) thatresulted in the isolation of a purer IgG preparation.

These methods, while laying the foundation for an entire industry ofplasma derived blood factors, were unable to provide IgG preparationshaving sufficiently high concentrations for the treatment of severalimmune-related diseases, including Kawasaki syndrome, immunethrombocytopenic purpura, and primary immune deficiencies. As such,additional methodologies employing various techniques, such as ionexchange chromatography, were developed to provide higher purity andhigher concentration IgG formulations. Hoppe et al. (Munch MedWochenschr 1967 (34): 1749-1752) and Falksveden (Swedish Patent No.348942) and Falksveden and Lundblad (Methods of Plasma ProteinFractionation 1980) were among the first to employ ion exchangechromatography for this purpose.

Various modern methods employ a precipitation step, such as caprylateprecipitation (Lebing et al., Vox Sang 2003 (84):193-201) and CohnFraction (I+)II+III ethanol precipitation (Tanaka et al., Braz J MedBiol Res 2000 (33) 37-30) coupled to column chromatography. Mostrecently, Teschner et al. (Vox Sang, 2007 (92):42-55) have described amethod for production of a 10% IVIG product in which cryo-precipitate isfirst removed from pooled plasma and then a modified Cohn-Oncley coldethanol fractionation is performed, followed by S/D treatment of theintermediate, ion exchange chromatography, nanofiltration, andoptionally ultrafiltration/diafiltration.

However, despite the improved purity, safety, and yield afforded bythese IgG manufacturing methods, highly concentrated IgG preparationssuitable for subcutaneous and/or intramuscular administration are stillneeded. The present invention fulfills these other needs and describesthe manufacturing method of a stable, highly purified, virusinactivated, ready-to-use product with high concentration of IgG.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides an aqueous compositioncomprising more than about 180 grams of protein per liter of thecomposition, and at least 95% of the protein is IgG, such as human IgG.In some embodiments, the composition is produced by a process leading toa product suitable for subcutaneous and intravenous administration, andcan be treated at elevated temperature in the final container toinactivate viruses regardless which concentration is adjusted between aconcentration range of 10 to 22% of protein. In some cases, the proteinconcentration in the composition is at or about 20% (w/v). In othercases, the composition may further comprise about 0.1-0.3 M glycine. Thecomposition of this invention may have varying pH, such as about 3-6, orabout 4-6.

In another aspect, this invention provides a method for preparing acomposition of concentrated IgG from plasma with the improvementcomprising the steps of: (1) concentrating protein in a plasmapreparation to at or about 5% (w/v) by ultrafiltration; and (2) furtherconcentrating the protein in the preparation to at or about 20% (w/v) bydiafiltration. At least 95% of the protein referred to in thecomposition is IgG, such as human IgG. In some embodiments, step (1) isperformed using an ultrafiltration membrane with a nominal molecularweight cut off (NMWCO) of 100 kDa or less. In other embodiments, step(2) is performed against a diafiltration buffer of glycine with a pH of4.2±0.1. The diafiltration buffer in some cases has 0.25 M glycine and apH of 4.0. In some particular embodiments, the protein concentrationafter step (2) is higher than 20% (w/v) and is subsequently adjusted toat or about 20% (w/v) with a diafiltration buffer.

In another aspect, the invention provides a method for preparing acomposition of concentrated IgG from plasma, comprising the steps of:

-   -   (1) separating liquid and precipitate from plasma by        centrifugation;    -   (2) mixing pre-cooled ethanol with the liquid from (1) to form a        mixture, which has an ethanol concentration of at or about 8%        (v/v);    -   (3) separating liquid and precipitate from the mixture of (2) by        centrifugation;    -   (4) adjusting pH and ethanol concentration of the liquid        from (3) to at or about 7.0 and 20-25% (v/v), respectively,        thereby forming a mixture;    -   (5) separating liquid and precipitate from the mixture of (4) by        centrifugation;    -   (6) resuspending the precipitate of (5) with a buffer at a ratio        of at or about 1 to 15 in weight to form a suspension;    -   (7) mixing silicon dioxide (SiO₂) with the suspension from (6)        and obtaining a filtrate by filtration;    -   (8) mixing a detergent and cold alcohol with the filtrate of (7)        and obtaining a precipitate by centrifugation;    -   (9) dissolving the precipitate in an aqueous solution comprising        a solvent or detergent and maintaining the solution for at least        60 minutes;    -   (10) passing the solution after (9) through a cation exchange        chromatography column and eluting proteins absorbed on the        column in an eluate;    -   (11) passing the eluate from (10) through an anion exchange        chromatography column to generate an effluent;    -   (12) passing the effluent through a nanofilter to generate a        nanofiltrate;    -   (13) passing the nanofiltrate through an ultrafiltration        membrane to generate an ultrafiltrate;    -   (14) diafiltrating the ultrafiltrate against a diafiltration        buffer to generate a solution having a protein concentration of        at or about 20% (w/v); and    -   (15) sterilizing the solution from (14) by filtering the        solution through a filter of 0.2 μm or less, thereby obtaining a        composition of concentrated IgG.

In some embodiments, step (2) is performed at a temperature of about −2to 0° C.; or the mixture of step (2) is mixed for at least 15 minutesand then maintained for at least 2 hours at a temperature of about −2 to0° C. In some embodiments, step (4) is mixed for at least 15 minutes andthen maintained for at least 8 hours at a temperature of at or about −7°C. In some embodiments, the suspension of step (6) is stirred for about40-160 minutes at a temperature of about 2° C. to 8° C. and a pH of ator about 5.0; or the silicon dioxide in step (7) is at a concentrationof 40 g/kg of the suspension of step (6) and the mixing is performed ata temperature of about 2 to 8° C. for at least about 50 minutes. In someembodiments, step (8) is performed at a temperature of about −5 to −10°C. In some embodiments, the solution of step (9) comprises 1.0% (v/v)Triton X-100, 0.3% (v/v) Tween-80, and 0.3% (v/v) Tri-(n-butyl)phosphate (TNBP). In some embodiments, the solution of step (9) ismaintained at a temperature of about 18 to 25° C. In some embodiments,the cation exchange chromatography column of step (10) is washed with a10 mM acetate buffer of pH 5.5±0.1 and eluted with a buffer of 35 mMmonobasic sodium phosphate, 10 mM Tris, pH 8.5±0.1, conductivity 5.0±0.2mS/cm. In some embodiments, the eluate from step (10) is adjusted to apH of 6.4±0.2 and conductivity of about 1.5 to 2.5 mS/cm prior to step(11). In some embodiments, the effluent of step (11) is passed through afilter of 0.2 μm or smaller pore size prior to step (12). In someembodiments, the ultrafiltrate of step (13) has a protein concentrationof at or about 5±1% (w/v). In other embodiments, the ultrafiltrationmembrane of step (13) has a nominal molecular weight cut off (NMWCO) of50 kDa or less. In some embodiments, the diafiltration buffer of step(14) is a 0.25 M glycine solution with a pH of 4.2±0.1. In otherembodiments, the solution from step (14) has a protein concentrationgreater than 20% (w/v) and is subsequently adjusted to at or about20.4±0.4% (w/v) with the diafiltration buffer. In some embodiments,steps (13) and (14) are performed at a temperature of about 2 to 8° C.In other embodiments, the preparation method further comprises a step ofdispensing the sterilized solution of step (15) into containers understerile conditions before the containers are sealed.

In other embodiments, the method described above may further comprise astep of storing the sealed containers at about 30 to 32° C. for about 21to 22 days; or the method may further comprise a formulation step torender the 20% IgG product as stable as the 10% state of the artintravenous formulation.

In yet another aspect, this invention provides an aqueous compositionthat is produced by the preparation method described above and comprisesat least 18% (w/v) immunoglobulin, for instance, at least 20% (w/v)immunoglobulin.

In a further aspect, the present invention provides a method fortreating a patient suffering from an immunodeficiency, an autoimmunedisease, or an acute infection, comprising administering to the patientan effective amount of the composition as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E are graphic illustrations of the newultrafiltration/diafiltration system.

FIG. 2 provides an overview of the downstream part of the manufacturingscheme for Subcuvia NG Solution starting with Step 10. As describedfurther in Example 5, viral inactivation by low pH treatment was assayedaccording to the sampling plan shown under process scheme B. Briefly, asample was drawn for BVDV and MMV testing after the virus spike, “SSM.”A second sample was drawn for BVDV and MMV testing after the virus spikeand then filtered using a 0.45 μm PVDF membrane, “SSM filt.” After pHadjustment and filtration in scheme B, a first sample was drawn for BVDVand MMV testing as soon as the temperature reached 29° C., “0 d.”Subsequent samples were drawn after storage for 7, 14, 20, and 27 daysat 30° C.±1° C., “7 d,” “14 d,” “20 d,” and “27 d,” respectively.Additional samples for bulk titration of BVDV were drawn after storagefor 20 and 27 days “20 d Bulk 0,” “20 d Bulk −0.5,” “20 d Bulk −1,” “27d Bulk 0,” “27 d Bulk −0.5,” and “27 d Bulk −1.”

FIG. 3 illustrates the sequence of virus spike, pH adjustments and 0.45mm filtration steps.

DEFINITIONS

An “antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof, whichspecifically bind and recognize an analyte (antigen). The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD, and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit is composed oftwo pairs of polypeptide chains, each pair having one “light” (about 25kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chaindefines a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The terms variable lightchain (V_(L)) and variable heavy chain (V_(H)) refer to these light andheavy chains respectively.

The term “ultrafiltration (UF)” encompasses a variety of membranefiltration methods in which hydrostatic pressure forces a liquid againsta semi-permeable membrane. Suspended solids and solutes of highmolecular weight are retained, while water and low molecular weightsolutes pass through the membrane. This separation process is often usedfor purifying and concentrating macromolecular (10³-10⁶ Da) solutions,especially protein solutions. A number of ultrafiltration membranes areavailable depending on the size of the molecules they retain.Ultrafiltration is typically characterized by a membrane pore sizebetween 1 and 1000 kDa and operating pressures between 0.01 and 10 bar,and is particularly useful for separating colloids like proteins fromsmall molecules like sugars and salts.

The term “diafiltration” is performed with the same membranes asultrafiltration and is a tangential flow filtration. Duringdiafiltration, buffer is introduced into the recycle tank while filtrateis removed from the unit operation. In processes where the product is inthe retentate (for example IgG), diafiltration washes components out ofthe product pool into the filtrate, thereby exchanging buffers andreducing the concentration of undesirable species.

As used herein, the word “about” denotes an approximate range of plus orminus 10% from a specified value. For instance, the language “about 20%”encompasses a range of 18-22%.

The term “mixing” describes an act of causing equal distribution of twoor more distinct compounds or substances in a solution or suspension byany form of agitation. Complete equal distribution of all ingredients ina solution or suspension is not required as a result of “mixing” as theterm is used in this application.

In this application, the term “solvent” encompasses any liquid substancecapable of dissolving or dispersing one or more other substances. Asolvent may be inorganic in nature, such as water, or it may be anorganic liquid, such as ethanol, acetone, methyl acetate, ethyl acetate,hexane, petrol ether, etc. As used in the term “solvent detergenttreatment,” solvent denotes an organic solvent (e.g., tri-N-butylphosphate), which is part of the solvent detergent mixture used toinactivate lipid-enveloped viruses in solution.

The term “detergent” is used in this application interchangeably withthe term “surfactant” or “surface acting agent.” Surfactants aretypically organic compounds that are amphiphilic, i.e., containing bothhydrophobic groups (“tails”) and hydrophilic groups (“heads”), whichrender surfactants soluble in both organic solvents and water. Asurfactant can be classified by the presence of formally charged groupsin its head. A non-ionic surfactant has no charge groups in its head,whereas an ionic surfactant carries a net charge in its head. Azwitterionic surfactant contains a head with two oppositely chargedgroups. Some examples of common surfactants include: Anionic (based onsulfate, sulfonate or carboxylate anions): perfluorooctanoate (PFOA orPFO), perfluorooctanesulfonate (PFOS), sodium dodecyl sulfate (SDS),ammonium lauryl sulfate, and other alkyl sulfate salts, sodium laurethsulfate (also known as sodium lauryl ether sulfate, or SLES), alkylbenzene sulfonate; cationic (based on quaternary ammonium cations):cetyl trimethylammonium bromide (CTAB) a.k.a. hexadecyl trimethylammonium bromide, and other alkyltrimethylammonium salts,cetylpyridinium chloride (CPC), polyethoxylated tallow amine (POEA),benzalkonium chloride (BAC), benzethonium chloride (BZT); Long chainfatty acids and their salts: including caprylate, caprylic acid,heptanoat, hexanoic acid, heptanoic acid, nanoic acid, decanoic acid,and the like; Zwitterionic (amphoteric): dodecyl betaine; cocamidopropylbetaine; coco ampho glycinate; nonionic: alkyl poly(ethylene oxide),alkylphenol poly(ethylene oxide), copolymers of poly(ethylene oxide) andpoly(propylene oxide) (commercially known as Poloxamers or Poloxamines),alkyl polyglucosides, including octyl glucoside, decyl maltoside, fattyalcohols (e.g., cetyl alcohol and oleyl alcohol), cocamide MEA, cocamideDEA, polysorbates (Tween 20, Tween 80, etc.), Triton detergents, anddodecyl dimethylamine oxide.

As used herein, the term “Intravenous IgG” or “IVIG” treatment refersgenerally to a therapeutic method of intravenously, subcutaneously, orintramuscularly administering a composition of IgG immunoglobulins to apatient for treating a number of conditions such as immune deficiencies,inflammatory diseases, and autoimmune diseases. The IgG immunoglobulinsare typically pooled and prepared from plasma. Whole antibodies orfragments can be used. IgG immunoglobulins can be formulated in higherconcentrations (e.g., greater than 10%) for subcutaneous administration,or formulated for intramuscular administration. This is particularlycommon for specialty IgG preparations which are prepared with higherthan average titres for specific antigens (e.g., Rho D factor, pertussistoxin, tetanus toxin, botulism toxin, rabies, etc.). For ease ofdiscussion, such subcutaneously or intramuscularly formulated IgGcompositions are also included in the term “IVIG” in this application.

By “therapeutically effective amount or dose” or “sufficient/effectiveamount or dose,” it is meant a dose that produces effects for which itis administered. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins; the disclosures of whichare incorporated by reference herein in their entireties for allpurposes).).

DETAILED DESCRIPTION OF THE INVENTION

As routinely practiced in modern medicine, sterilized preparations ofconcentrated immunoglobulins (especially IgGs) are used for treatingmedical conditions that fall into three main classes: immunedeficiencies, inflammatory and autoimmune diseases, and acuteinfections. One commonly used IgG product, intravenous immunoglobulin orIVIG, is formulated for intravenous administration, for example, at a10% concentration. Concentrated immunoglobulins may also be formulatedfor subcutaneous or intramuscular administration, for example at orabout a 20% concentration.

In one aspect, the present invention relates to a new and improvedmethod for producing highly purified and highly concentratedimmunoglobulin compositions from pooled plasma. Compared to thepreviously used IgG purification and concentration methods, theinventors have incorporated ultrafiltration and formulation steps, whichresult in higher IgG concentration without significant IgG loss andmaintain low pH in the final formulation. Typically, the products have aprotein concentration of at least 18% weight/volume (w/v), of which vastmajority (typically no less than 95%) is IgG, and a pH in the range ofpH 3-6, which facilitates inactivation of pathogens such as viruses thatmay be present in the plasma. Due to their high IgG concentration andtherefore reduced volume in administration, the products of thisinvention are suitable for subcutaneous and/or intramuscularadministration. In some embodiments, the IgG products have a viscosityno greater than 18 mPascal-second and may therefore be suitable forintravenous administration as well. Due to the possibility to combinequality attributes for intravenous products with the required highconcentration for subcutaneous and intramuscular products, simpledilution can also enable intravenous administration. A further advantageof the IgG composition of this invention is that they possess excellentstability during storage.

In certain aspects, the present invention provides methods for preparinga highly concentrated IgG preparation with a final proteinconcentrations of greater than about 17% and an IgG purity of at leastabout 95%. In certain embodiments, the preparation has an extendedstability and is formulated for intravenous, subcutaneous, and/orintramuscular administration.

In another aspect, the present invention provides pharmaceuticalcompositions and formulations of IgG compositions prepared according tothe improved manufacturing methodologies provided herein. In certainembodiments, these compositions and formulations provide improvedproperties as compared to other IVIG compositions currently on themarket. For example, in certain embodiments, the compositions andformulations provided herein are stable for an extended period of time.In another embodiment, compositions and formulations provided hereinhave a higher IgG concentration as compared to other IVIG compositionscurrently on the market. In yet other embodiments, compositions andformulations provided herein have a higher IgG concentration and arestable for an extended period of time.

In yet another aspect, the present invention provides method fortreating immune deficiencies, inflammatory and autoimmune diseases, andacute infections comprising the administration of an IgG compositionprepared using the improved methods provided herein.

I. Producing a Concentrated, Purified IgG Preparation

IVIG compositions comprising whole antibodies have been described forthe treatment of certain autoimmune conditions. (See, e.g., U.S. PatentPublication US 2002/0114802, US 2003/0099635, and US 2002/0098182.) TheIVIG compositions disclosed in these references include polyclonalantibodies.

Generally, immunoglobulin preparations according to the presentinvention can be prepared from any suitable starting materials, forexample, recovered plasma or source plasma. In a typical example, bloodor plasma is collected from healthy donors. Usually, the blood iscollected from the same species of animal as the subject to which theimmunoglobulin preparation will be administered (typically referred toas “homologous” immunoglobulins). The immunoglobulins are isolated fromthe blood by suitable procedures, such as, for example, precipitation(alcohol fractionation or polyethylene glycol fractionation),chromatographic methods (ion exchange chromatography, affinitychromatography, immunoaffinity chromatography) ultracentrifugation, andelectrophoretic preparation, and the like. (See, e.g., Cohn et al., J.Am. Chem. Soc. 68:459-75 (1946); Oncley et al., J. Am. Chem. Soc.71:541-50 (1949); Barundern et al., Vox Sang. 7:157-74 (1962); Koblet etal., Vox Sang. 13:93-102 (1967); U.S. Pat. Nos. 5,122,373 and 5,177,194;the disclosures of which are incorporated by reference herein in theirentireties for all purposes).

Unlike the methods described above, in one aspect the present inventionprovides methods of preparing concentrated IgG compositions that utilizea cryo-poor starting material. Generally, the methods provided hereinutilize both modified Cohn-Oncley alcohol fractionation steps and ionexchange chromatography to provide superior IgG yields, whilemaintaining the same, if not improved, quality as found in currentlyavailable commercial IVIG preparations.

In many cases, immunoglobulin is prepared from gamma globulin-containingproducts produced by the alcohol fractionation and/or ion exchange andaffinity chromatography methods well known to those skilled in the art.Purified Cohn Fraction II is commonly used. The starting Cohn FractionII paste is typically at or about 95 percent IgG and is comprised of thefour IgG subtypes. The different subtypes are present in Fraction II inapproximately the same ratio as they are found in the pooled humanplasma from which they are obtained. The Fraction II is further purifiedbefore formulation into an administrable product. For example, theFraction II paste can be dissolved in a cold purified aqueous alcoholsolution and impurities removed via precipitation and filtration.Following the final filtration, the immunoglobulin suspension can bedialyzed or diafiltered (e.g., using ultrafiltration membranes having anominal molecular weight limit of less than or equal to 100,000 daltons)to remove the alcohol. The solution can be concentrated or diluted toobtain the desired protein concentration and can be further purified bytechniques well known to those skilled in the art.

Preparative steps can be used to enrich a particular isotype or subtypeof immunoglobulin. For example, protein A, protein G or protein Hsepharose chromatography can be used to enrich a mixture ofimmunoglobulins for IgG, or for specific IgG subtypes. (See generallyHarlow and Lane, Using Antibodies, Cold Spring Harbor Laboratory Press(1999); Harlow and Lane, Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory Press (1988); U.S. Pat. No. 5,180,810.)

As will be described in detail below, the high concentration IgGproducts of this invention are produced by a process having many of thesame or similar steps as in the process of producing IVIG. Theadditional steps of ultrafiltration/diafiltration using open channelmembranes with a specifically designed post-wash and formulation nearthe end the production process render the resulting IgG compositionsabout twice as high in protein concentration (200 mg/mL) compared tostate of the art IVIGs (e.g., GAMMAGARD® LIQUID) without affecting yieldand storage stability. With most of the commercial availableultrafiltration membranes a concentration of 200 mg/mL IgG cannot bereached without major protein losses. These membranes will be blockedearly and therefore adequate post-wash is difficult to achieve.Therefore open channel membrane configurations have to be used. Evenwith open channel membranes, a specifically designed post-wash procedurehas to be used to obtain the required concentration without significantprotein loss (less than 2% loss). Even more surprising is the fact thatthe higher protein concentration of 200 mg/mL does not effect the virusinactivation capacity of the low pH storage step. The general process ofproducing the high concentration IgG composition includes the followingsteps:

A. Separation of CryoPrecipitates

The purification process typically starts with thawing previously frozenpooled plasma, already checked for safety and quality considerations.Thawing is typically carried out at a temperature no higher than 6° C.Centrifugation or filtration is then performed in the cold to separatesolid and liquid upon plasma being thawed, usually at the same lowtemperature as thawing. The liquid portion (also referred to as“cryo-poor plasma,” after cold-insoluble proteins removed bycentrifugation or filtration from fresh thawed plasma) is then processedin the next step. Various additional steps can be taken at this juncturefor the isolation of factor eight inhibitor bypass activity (FEIBA),Factor IX-complex, Factor VII-concentrate, or Antithrombin III-complex,which are described in detail in Example 1.

B. Obtain Supernatant of Fractionation I

In this step, cyro-poor plasma is typically cooled to at or about 0±1°C., and its pH is adjusted to at or about 7.0. In certain embodiments,the pH is adjusted to between about 7.0 and about 7.5, preferablybetween at or about 7.1 and at or about 7.3, most preferably at or about7.2. In one embodiment, the pH is adjusted to at or about 7.0. Inanother embodiment, the pH is adjusted to at or about 7.1. In anotherembodiment, the pH is adjusted to at or about 7.2. In anotherembodiment, the pH is adjusted to at or about 7.3. In anotherembodiment, the pH is adjusted to at or about 7.4. In anotherembodiment, the pH is adjusted to at or about 7.5. Pre-cooled ethanol isthen added while the plasma is being stirred to a target concentrationof ethanol at 8% v/v. At the same time the temperature is furtherlowered to between about −4 and about 0° C., preferably about −2° C., toprecipitate contaminants such as α₂-macroglobulin, β_(1A)- andβ_(1C)-globulin, fibrinogen, and Factor VIII. Typically, theprecipitation event will include a hold time of at least about 1 hour,although shorter or longer hold times may also be employed.Subsequently, the supernatant (Supernatant I), ideally containing theentirety of the IgG content present in the cryo-poor plasma, is thencollected by centrifugation, filtration, or another suitable method.

C. Precipitate of Fractionation II+III

To further enrich the IgG content and purity of the fractionation,Supernatant I is subjected to a second precipitation step. Generally,the pH of the solution is adjusted to a pH of between about 6.8 andabout 7.2, preferably at or about 7.0. Alcohol, preferably ethanol, isthen added to the solution while being stirred to a final concentrationof between about 20% and about 25% (v/v). In one embodiment, the finalconcentration of alcohol is at or about 20%. In another embodiment, thefinal alcohol concentration is at or about 21%. In another embodiment,the final alcohol concentration is at or about 22%. In anotherembodiment, the final alcohol concentration is at or about 23%. Inanother embodiment, the final alcohol concentration is at or about 24%.In another embodiment, the final alcohol concentration is at or about25%. The liquid portion, also referred to as Fraction II+IIIsupernatant, can be further processed to extract Factor V. Theprecipitate from this step is processed further in the next step. In oneembodiment, steps B and C can also be performed together.

D. Extraction from Fractionations II and III Precipitate

A cold extraction buffer is used to resuspend the Fraction II+IIIprecipitate at a typical ratio of 1 part of precipitate in 15 parts ofextraction buffer. An exemplary extraction buffer contains 5 mMmonobasic sodium phosphate and 5 mM acetate, and has a pH at or about4.5±0.2 and conductivity of at or about 0.7 to 0.9 mS/cm. In oneembodiment, the conductivity of the extraction buffer is at or about 0.7mS/cm. In another embodiment, the conductivity of the extraction bufferis at or about 0.8 mS/cm. In yet another embodiment, the conductivity ofthe extraction buffer is at or about 0.9 mS/cm. The extraction processis performed at a temperature of at or about 2 to 8° C.

Other suitable re-suspension ratios may be used, for example from about1:8 to about 1:30, or from about 1:10 to about 1:20, or from about 1:12to about 1:18, or from about 1:13 to about 1:17, or from about 1:14 toabout 1:16. In certain embodiments, the re-suspension ratio may be at orabout 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18,1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30,or higher.

Suitable solutions for the extraction of the II+III precipitate willgenerally have a pH between about 4.0 and about 5.5. In certainembodiments, the solution will have a pH between about 4.0 and about5.0. In another embodiment, the solution will have a pH between about4.5 and about 5.0. In other embodiments, the extraction solution willhave a pH of about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,5.0, 5.1, 5.2, 5.3, 5.4, or 5.5. In one embodiment, the pH of theextraction buffer will be at or about 4.5. In another embodiment, the pHof the extraction buffer will be at or about 4.6. In another embodiment,the pH of the extraction buffer will be at or about 4.7. In anotherembodiment, the pH of the extraction buffer will be at or about 4.8. Inanother embodiment, the pH of the extraction buffer will be at or about4.9. In another embodiment, the pH of the extraction buffer will be ator about 5.0.

The extraction buffer will preferably have a conductivity of from about0.5 mS-cm⁻¹ to about 2.0 mS·cm⁻¹. For example, in certain embodiments,the conductivity of the extraction buffer will be at or about 0.5mS·cm⁻¹, or at or about 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, or about 2.0 mS-cm⁻¹. One of ordinary skill inthe art will know how to generate extraction buffers having anappropriate conductivity.

E. Fumed Silica Treatment and Filtration

In some embodiment, fumed silica (e.g., Aerosil 380 or equivalent) isadded to the suspension from the last step to a concentration of at orabout 40 g/kg of suspension, or equivalent to 1.8 g/liter of cryo-poorplasma. Mixing takes place at about 2 to 8° C. for at least 50 to 70minutes. In some cases, filter aid (e.g., Hyflo-Supper-Cel from WorldMinerals, used at a concentration of at or about 0.5 kg/kg ofsuspension) is added to facilitate the subsequent step of liquid/solidseparation by filtration. The extraction buffer is used for post-washingof the filter press. The filtration process is maintained at atemperature of about 2 to 8° C.

F. Fractionation of Precipitate G

Filtrate from the last step is then mixed with polysorbate-80 to aconcentration of at or about 0.2% w/v with stirring for at least 30minutes at a temperature of about 2° C. to 8° C. Sodium citratedehydrate is then mixed into the solution at or about 8 g/liter foranother 30 minutes of stirring at a temperature of about 2° C. to 8° C.The solution's pH is then adjusted to at or about 7.0±0.1. In certainembodiments, the pH is adjusted with either sodium hydroxide or aceticacid. Cold alcohol is then added to the solution to a concentration ofat or about 25% v/v, and a precipitation step similar to Cohn II isperformed.

G. Suspension of Precipitate G

The precipitate from the last step is dissolved and filtered with adepth filter of a nominal pore size of 0.2 μm (e.g., Cuno VR06 filter orequivalent) to obtain a clear filtrate. In another embodiment, theprecipitate is dissolved and then centrifuged to recover a clarifiedsupernatant.

H. Solvent and Detergent Treatment

The filtrate from the last step is used for the solvent-/detergenttreatment. A typical solvent/detergent treatment mixture comprises 1.0%(v/v) Triton X-100, 0.3% (v/v) Tween-80, and 0.3% (v/v) TNBP, and themixture is typically held at a temperature of about 18° C. to 25° C. forat least 60 minutes. Methods for the detergent treatment of plasmaderived fractions are well known in the art. Generally, any standardnon-ionic detergent treatment may be used in conjunction with themethods provided herein.

I. Cation Exchange Chromatographv

In order to further purify and concentrate IgG from the S/D treated PptGfiltrate, cation exchange and/or anion exchange chromatography can beemployed. Methods for purifying and concentrating IgG using ion exchangechromatography are well known in the art. For example, U.S. Pat. No.5,886,154 describes a method in which a Fraction II+III precipitate isextracted at low pH (between about 3.8 and 4.5), followed byprecipitation of IgG using caprylic acid, and finally implementation oftwo anion exchange chromatography steps. U.S. Pat. No. 6,069,236describes a chromatographic IgG purification scheme that does not relyon alcohol precipitation at all. PCT Publication No. WO 2005/073252describes an IgG purification method involving the extraction of aFraction II+III precipitate, caprylic acid treatment, PEG treatment, anda single anion exchange chromatography step. U.S. Pat. No. 7,186,410describes an IgG purification method involving the extraction of eithera Fraction I+II+III or a Fraction II precipitate followed by a singleanion exchange step performed at an alkaline pH. U.S. Pat. No. 7,553,938describes a method involving the extraction of either a FractionI+II+III or a Fraction II+III precipitate, caprylate treatment, andeither one or two anion exchange chromatography steps. U.S. Pat. No.6,093,324 describes a purification method comprising the use of amacroporous anion exchange resin operated at a pH between about 6.0 andabout 6.6. U.S. Pat. No. 6,835,379 describes a purification method thatrelies on cation exchange chromatography in the absence of alcoholfractionation.

In one embodiment, the solvent/detergent containing protein solutionfrom the last step is then passed through a cation exchange column toremove the solvent and detergent. After washing out of SD reagents, theabsorbed proteins are then eluted with high pH elution buffer. In oneembodiment, the elution buffer will have a pH of between about 7.5 andabout 9.5. In another embodiment, the elution buffer will have a pH ofbetween about 8.0 and about 9.0. In a preferred embodiment, the elutionbuffer will have a pH of at or about 8.5±0.1.

J. Anion Exchange Chromatographv

The eluate from the last step is adjusted to pH 6 and diluted to theappropriate conductivity for the following equilibrated anion exchangecolumn. The column flow-through during loading and washing is collectedfor further processing.

K. Nanofiltration

In order to further reduce the viral load of the IgG compositionprovided herein, the anion exchange column effluent may be nanofilteredusing a suitable nanofiltration device. In certain embodiments, thenanofiltration device will have a mean pore size of between about 15 nmand about 200 nm. Examples of nanofilters suitable for this use include,without limitation, DVD, DV 50, DV 20 (Pall), Viresolve NFP, ViresolveNFR (Millipore), Planova 15N, 20N, 35N, and 75N (Planova). In a specificembodiment, the nanofilter may have a mean pore size of between about 15nm and about 72 nm, or between about 19 nm and about 35 nm, or of at orabout 15 nm, 19 nm, 35 nm, or 72 nm. In a preferred embodiment, thenanofilter will have a mean pore size of at or about 35 nm, such as anAsahi PLANOVA 35N filter or equivalent thereof.

L. Ultrafiltration and Diafiltration

Subsequent to nanofiltration, the filtrate is further concentrated to aprotein concentration of 5±1% w/v by ultrafiltration. In some examples,the ultrafiltration is carried out in a cassette with an open channelscreen and the ultrafiltration membrane has a nominal molecular weightcut off (NMWCO) of 50 kDa or less.

In one embodiment, the nanofiltrate may be concentrated byultrafiltration to a protein concentration of between about 2% and about10% (w/v). In certain embodiments, the ultrafiltration is carried out ina cassette with an open channel screen and the ultrafiltration membranehas a nominal molecular weight cut off (NMWCO) of less than about 100kDa or less than about 90, 80, 70, 60, 50, 40, 30, or fewer kDa. In apreferred embodiment, the ultrafiltration membrane has a NMWCO of nomore than 50 kDa.

Upon completion of the ultrafiltration step, the concentrate may furtherbe concentrated via diafiltration against a solution suitable forintravenous or intramuscular administration. In certain embodiments, thediafiltration solution may comprise a stabilizing and/or bufferingagent. In a preferred embodiment, the stabilizing and buffering agent isglycine at an appropriate concentration, for example between about 0.20M and about 0.30M, or between about 0.22M and about 0.28M, or betweenabout 0.24M and about 0.26 mM, or at a concentration of at or about 2.0,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0. In a preferredembodiment, the diafiltration buffer contains at or about 0.25 Mglycine.

In a preferred embodiment, upon completion of the ultrafiltration step,the concentrate is diafiltered against a 0.25 M glycine solution with alow pH. Typically, the minimum exchange volume is 6 times of theoriginal concentrate volume, and the solution is concentrated to aprotein concentration of more than 20% w/v. At the end of thediafiltration and concentration process, the pH of the solution istypically between 4.4 to 4.9.

Typically, the minimum exchange volume is at least about 3 times theoriginal concentrate volume or at least about 4, 5, 6, 7, 8, 9, or moretimes the original concentrate volume. The IgG solution may beconcentrated to a final protein concentration of between about 5% andabout 22% (w/v), or between about 10% and about 22% (w/v), or betweenabout 15% and about 22% (w/v), or between about 18% and about 22% (w/v),or between about 20% and about 22%, or to a final concentration of at orabout 5%, or 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, or higher. In a preferred embodiment, the IgGsolution will be concentrated to final protein concentration of at orbetween about 20% and at or between 22%. Typically, at the end of theconcentration process, the pH of the solution will be between about 4.6to 5.1.

FIGS. 1A-1E illustrate an exemplary method forultrafiltration/diafiltration according to an embodiment of thedisclosure. First, a sample (e.g., a nanofiltrate) is concentrated to afirst concentration to form a first concentrate. The first concentrateis diafiltered against a diafiltration buffer to form a diafiltrate. Thediafiltrate is then concentrated to a protein value of greater than 22%to form a second concentrate. These steps are performed in a firstultra-/diafiltration system including a first batch tank and a firstmembrane, as shown in FIG. 1A. In some embodiments, the first batch tankis designed to hold small volumes, ideally by a conical bottom.

The second concentrate is then emptied from the firstultra-/diafiltration system, e.g., into a bulk tank, as shown in FIG.1B. The first ultra-/diafiltration system is then post-washed byre-circulation of a post wash buffer through the firstultra-/diafiltration system. The post-wash is transferred to a second,smaller, ultra-/diafiltration system that includes a second batch tankand a second membrane, as shown in FIG. 1C. The second membrane is thesame type of membrane as used in the first ultra-/diafiltration system.

The post-wash is then concentrated in the second ultra-/diafiltrationsystem to form a second concentrate, as shown in FIG. 1D. The secondconcentrate is then combined with the first concentrate, e.g., in thebulk tank, as shown in FIG. 1E. The second ultra-/diafiltration systemcan be post-washed, and the post-wash used to adjust the final proteinconcentration of the combined bulk solution, e.g., to 20.4%±0.4 w/v, notshown. The final bulk may be sterile filtered and filled into a finalcontainer.

M. Formulation

Upon completion of the diafiltration step, the protein concentration ofthe solution is adjusted to with the diafiltration buffer to a finalconcentration of between about 5% and about 20% (w/v), or between about10% and about 20% (w/v), or between about 15% and about 20% (w/v), orbetween about 18% and about 20% (w/v), or to a final concentration ofabout 5%, or 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, or 20%. In a preferred embodiment, the final proteinconcentration of the solution is at or between about 19% and at or about21%. In a preferred embodiment, upon completion of diafiltration, theprotein concentration of the solution is adjusted to just over 20% w/v,e.g., at or about 20.4±04% w/v, with the diafiltration buffer.

N. Further Sterilization

The formulated bulk solution is further sterilized by first filteringthrough a membrane filter with an absolute pore size of 0.2 micron orless. Then the solution is aseptically dispensed into final containersfor proper sealing, with samples taken for testing. The final step isstoring the sealed containers at 30 to 32° C. for an extended timeperiod, e.g., 21 to 22 days.

II. Concentrated IgG Compositions

In one aspect, the present invention relates to aqueous IgG compositionsprepared by the methods provided herein. Generally, the IgG compositionsprepared by the novel methods described herein will have high IgGcontent and purity. For example, IgG compositions provided herein mayhave a protein concentration of at least about 15% (w/v) and an IgGcontent of greater than 90% purity. These high purity IgG compositionsare suitable for therapeutic administration, e.g., for subcutaneousand/or intramuscular administration. In one embodiment, the IgGcompositions provided herein are suitable for intravenousadministration, for example by diluting prior to administration. In oneembodiment, the concentration of IgG is at or about 20% and is used forsubcutaneous or intramuscular administration.

In one embodiment, the present invention provides an aqueous IgGcomposition prepared by a method comprising the steps of:

-   -   (1) separating liquid and precipitate from plasma by        centrifugation;    -   (2) mixing pre-cooled ethanol with the liquid from (1) to form a        mixture, which has an ethanol concentration of at or about 8%        (v/v);    -   (3) separating liquid and precipitate from the mixture of (2) by        centrifugation;    -   (4) adjusting pH and ethanol concentration of the liquid        from (3) to at or about 7.0 and 20-25% (v/v), respectively,        thereby forming a mixture;    -   (5) separating liquid and precipitate from the mixture of (4) by        centrifugation;    -   (6) resuspending the precipitate of (5) with a buffer at a ratio        of about 1 to 15 in weight to form a suspension;    -   (7) mixing silicon dioxide (SiO2) with the suspension from (6)        and obtaining a filtrate by filtration;    -   (8) mixing a detergent and cold alcohol with the filtrate of (7)        and obtaining a precipitate by centrifugation;    -   (9) dissolving the precipitate in an aqueous solution comprising        a solvent or detergent and maintaining the solution for at least        60 minutes;    -   (10) passing the solution after (9) through a cation exchange        chromatography column and eluting proteins absorbed on the        column in an eluate;    -   (11) passing the eluate from (10) through an anion exchange        chromatography column to generate an effluent;    -   (12) passing the effluent through a nanofilter to generate a        nanofiltrate;    -   (13) passing the nanofiltrate through an ultrafiltration        membrane to generate an ultrafiltrate;    -   (14) diafiltrating the ultrafiltrate against a diafiltration        buffer to generate a solution having a protein concentration of        at or about 20% (w/v); and    -   (15) sterilizing the solution from (14) by filtering the        solution through a filter of 0.2 μm or less, thereby obtaining a        composition of concentrated IgG.

In one embodiment, the present invention provides aqueous IgGcompositions comprising a protein concentration of between about 150 g/Land about 250 g/L. In certain embodiments, the protein concentration ofthe IgG composition is between about 175 g/L and about 225 g/L, orbetween about 200 g/L and about 225 g/L, or any suitable concentrationwithin these ranges, for example at or about, 150 g/L, 155 g/L, 160 g/L,165 g/L, 170 g/L, 175 g/L, 180 g/L, 185 g/L, 190 g/L, 195 g/L, 200 g/L,205 g/L, 210 g/L, 215 g/L, 220 g/L, 225 g/L, 230 g/L, 235 g/L, 240 g/L,245 g/L, 250 g/L, or higher. In a preferred embodiment, the aqueous IgGcomposition comprises a protein concentration of at or about 200 g/L. Ina particularly preferred embodiment, the aqueous IgG compositioncomprises a protein concentration of at or about 204 g/L.

The methods provided herein allow for the preparation of IgGcompositions having very high levels of purity. For example, in oneembodiment, at least about 95% of the total protein in a compositionprovided herein will be IgG. In other embodiments, at least about 96% ofthe protein is IgG, or at least about 97%, 98%, 99%, 99.5%, or more ofthe total protein of the composition will be IgG.

Similarly, the methods provided herein allow for the preparation of IgGcompositions which containing extremely low levels of contaminatingagents. For example, in certain embodiments, IgG compositions areprovided that contain less than about 100 mg/L IgA. In otherembodiments, the IgG composition will contain less than about 50 mg/LIgA, preferably less than about 35 mg/L IgA, most preferably less thanabout 20 mg/L IgA.

III. Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticalcompositions and formulations comprising purified IgG prepared by themethods provided herein. Generally, the IgG pharmaceutical compositionsand formulations prepared by the novel methods described herein willhave high IgG content and purity. For example, IgG pharmaceuticalcompositions and formulations provided herein may have a proteinconcentration of at least about 15% (w/v) and an IgG content of greaterthan 90% purity. These high purity IgG compositions are suitable fortherapeutic administration, e.g., for subcutaneous and/or intramuscularadministration. In one embodiment, the IgG compositions provided hereinare suitable for intravenous administration, for example by dilutingprior to administration. In one embodiment, the concentration of IgG isat or about 20% and is used for subcutaneous or intramuscularadministration.

In one embodiment, the pharmaceutical compositions provided herein areprepared by formulating an aqueous IgG composition isolated using amethod provided herein. Generally, the formulated composition will havebeen subjected to at least one, preferably at least two, most preferablyat least three, viral inactivation or removal steps. Non-limitingexamples of viral inactivation or removal steps that may be employedwith the methods provided herein include, solvent detergent treatment(Horowitz et al., Blood Coagul Fibrinolysis 1994 (5 Suppl 3):S21-S28 andKreil et al., Transfusion 2003 (43): 1023-1028, both of which are hereinexpressly incorporated by reference in their entirety for all purposes),nanofiltration (Hamamoto et al., Vox Sang 1989 (56) 230-236 and Yuasa etal., J Gen Virol. 1991 (72 (pt 8)):2021-2024, both of which are hereinexpressly incorporated by reference in their entirety for all purposes),and low pH incubation at high temperatures (Kempf et al., Transfusion1991 (31)423-427 and Louie et al., Biologicals 1994 (22):13-19).

In certain embodiments, pharmaceutical formulations are provided havingan IgG content of between about 175 g/L IgG and about 225 g/L IgG.Generally, these IVIG formulations are prepared by isolating an IgGcomposition from plasma using a method described herein, concentratingthe composition, and formulating the concentrated composition in asolution suitable for intravenous administration. The IgG compositionsmay be concentrated using any suitable method known to one of skill inthe art. In one embodiment, the composition is concentrated byultrafiltration/diafiltration. In some embodiments, the ultrafiltrationdevice used to concentrate the composition will employ anultrafiltration membrane having a nominal molecular weight cut off(NMWCO) of less than about 100 kDa or less than about 90, 80, 70, 60,50, 40, 30, or fewer kDa. In a preferred embodiment, the ultrafiltrationmembrane has a NMWCO of no more than 50 kDa. Buffer exchange may beachieved using any suitable technique known to one of skill in the art.In a specific embodiment, buffer exchange is achieved by diafiltration.

In a specific embodiment, a pharmaceutical composition of IgG isprovided, wherein the IgG composition was purified from plasma using amethod comprising the steps of

-   -   (1) separating liquid and precipitate from plasma by        centrifugation;    -   (2) mixing pre-cooled ethanol with the liquid from (1) to form a        mixture, which has an ethanol concentration of at or about 8%        (v/v);    -   (3) separating liquid and precipitate from the mixture of (2) by        centrifugation;    -   (4) adjusting pH and ethanol concentration of the liquid        from (3) to at or about 7.0 and 20-25% (v/v), respectively,        thereby forming a mixture;    -   (5) separating liquid and precipitate from the mixture of (4) by        centrifugation;    -   (6) resuspending the precipitate of (5) with a buffer at a ratio        of about 1 to 15 in weight to form a suspension;    -   (7) mixing silicon dioxide (SiO2) with the suspension from (6)        and obtaining a filtrate by filtration;    -   (8) mixing a detergent and cold alcohol with the filtrate of (7)        and obtaining a precipitate by centrifugation;    -   (9) dissolving the precipitate in an aqueous solution comprising        a solvent or detergent and maintaining the solution for at least        60 minutes;    -   (10) passing the solution after (9) through a cation exchange        chromatography column and eluting proteins absorbed on the        column in an eluate;    -   (11) passing the eluate from (10) through an anion exchange        chromatography column to generate an effluent;    -   (12) passing the effluent through a nanofilter to generate a        nanofiltrate;    -   (13) passing the nanofiltrate through an ultrafiltration        membrane to generate an ultrafiltrate;    -   (14) diafiltrating the ultrafiltrate against a diafiltration        buffer to generate a solution having a protein concentration of        about 20% (w/v); and    -   (15) sterilizing the solution from (14) by filtering the        solution through a filter of 0.2 μm or less, thereby obtaining a        composition of concentrated IgG.

In one embodiment, the present invention provides pharmaceutical IgGcompositions comprising a protein concentration of between about 150 g/Land about 250 g/L. In certain embodiments, the protein concentration ofthe IgG composition is between about 175 g/L and about 225 g/L, orbetween about 200 g/L and about 225 g/L, or any suitable concentrationwithin these ranges, for example at or about, 150 g/L, 155 g/L, 160 g/L,165 g/L, 170 g/L, 175 g/L, 180 g/L, 185 g/L, 190 g/L, 195 g/L, 200 g/L,205 g/L, 210 g/L, 215 g/L, 220 g/L, 225 g/L, 230 g/L, 235 g/L, 240 g/L,245 g/L, 250 g/L, or higher. In a preferred embodiment, the aqueous IgGcomposition comprises a protein concentration of at or about 200 g/L. Ina particularly preferred embodiment, the aqueous IgG compositioncomprises a protein concentration of at or about 204 g/L.

The methods provided herein allow for the preparation of IgGpharmaceutical compositions having very high levels of purity. Forexample, in one embodiment, at least about 95% of the total protein in acomposition provided herein will be IgG. In other embodiments, at leastabout 96% of the protein is IgG, or at least about 97%, 98%, 99%, 99.5%,or more of the total protein of the composition will be IgG.

Similarly, the methods provided herein allow for the preparation of IgGpharmaceutical compositions which containing extremely low levels ofcontaminating agents. For example, in certain embodiments, IgGcompositions are provided that contain less than about 100 mg/L IgA. Inother embodiments, the IgG composition will contain less than about 50mg/L IgA, preferably less than about 35 mg/L IgA, most preferably lessthan about 20 mg/L IgA.

The pharmaceutical compositions provided herein will typically compriseone or more buffering agents or pH stabilizing agents suitable forintravenous administration. Non-limiting examples of buffering agentssuitable for formulating an IgG composition provided herein includeglycine, citrate, phosphate, acetate, glutamate, tartrate, benzoate,lactate, histidine or other amino acids, gluconate, malate, succinate,formate, propionate, carbonate, or any combination thereof adjusted toan appropriate pH. Generally, the buffering agent will be sufficient tomaintain a suitable pH in the formulation for an extended period oftime. In a preferred embodiment, the buffering agent is glycine.

In some embodiments, the concentration of buffering agent in theformulation will be between about 100 mM and about 400 mM, preferablybetween about 150 mM and about 350 mM, more preferably between about 150mM and about 300 mM, most preferably between about 175 mM and about 225mM. In a particularly preferred embodiment, the concentrated IgGcomposition will comprise between about 150 mM and about 250 mM glycine,most preferably about 200 mM glycine. In another preferred embodiment,the concentrated IgG composition will contain at or about 250 mMglycine.

In certain embodiments, the pH of the formulation will be between about4.1 and about 5.6, preferably between about 4.4 and about 5.3, mostpreferably between about 4.6 and about 5.1. In particular embodiments,the pH of the formulation may be about 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, or 5.6.

In some embodiments, the pharmaceutical compositions provided herein mayoptionally further comprise an agent for adjusting the osmolarity of thecomposition. Non-limiting examples of osmolarity agents includemannitol, sorbitol, glycerol, sucrose, glucose, dextrose, levulose,fructose, lactose, polyethylene glycols, phosphates, sodium chloride,potassium chloride, calcium chloride, calcium gluconoglucoheptonate,dimethyl sulfone, and the like.

Typically, the formulations provided herein will have osmolarities thatare comparable to physiologic osmolarity, about 285 to 295 mOsmol/kg(Lacy et al., Drug Information Handbook-Lexi-Comp 1999:1254. In certainembodiments, the osmolarity of the formulation will be between about 200mOsmol/kg and about 350 mOsmol/kg, preferably between about 240 andabout 300 mOsmol/kg. In particular embodiments, the osmolarity of theformulation will be about 200 mOsmol/kg, or 210 mOsmol/kg, 220mOsmol/kg, 230 mOsmol/kg, 240 mOsmol/kg, 245 mOsmol/kg, 250 mOsmol/kg,255 mOsmol/kg, 260 mOsmol/kg, 265 mOsmol/kg, 270 mOsmol/kg, 275mOsmol/kg, 280 mOsmol/kg, 285 mOsmol/kg, 290 mOsmol/kg, 295 mOsmol/kg,300 mOsmol/kg, 310 mOsmol/kg, 320 mOsmol/kg, 330 mOsmol/kg, 340mOsmol/kg, 340 mOsmol/kg, or 350 mOsmol/kg.

The IgG formulations provided herein are generally stable in liquid formfor an extended period of time. In certain embodiments, the formulationsare stable for at least about 3 months at room temperature, or at leastabout 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, or 24 months at room temperature. The formulation will alsogenerally be stable for at least about 18 months under refrigeratedconditions (typically between about 2° C. and about 8° C.), or for atleast about 21, 24, 27, 30, 33, 36, 39, 42, or 45 months underrefrigerated conditions.

IV. Administration of the IgG Preparation

As routinely practiced in the modern medicine, sterilized preparationsof concentrated immunoglobulins (especially IgGs) are used for treatingmedical conditions that fall into these three main classes: immunedeficiencies, inflammatory and autoimmune diseases, and acuteinfections. These IgG preparations may also be useful for treatingmultiple sclerosis (especially relapsing-remitting multiple sclerosis orRRMS), Alzheimer's disease, and Parkinson's disease. The purified IgGpreparation of this invention is suitable for these purposes, as well asother clinically accepted uses of IgG preparations.

The FDA has approved the use of IVIG to treat various indications,including allogeneic bone marrow transplant, chronic lymphocyticleukemia, idiopathic thrombocytopenic purpura (ITP), pediatric HIV,primary immunodeficiencies, Kawasaki disease, chronic inflammatorydemyelinating polyneuropathy (CIDP), and kidney transplant with a highantibody recipient or with an ABO incompatible donor. In certainembodiments, the IVIG compositions provided herein are useful for thetreatment or management of these diseases and conditions.

Furthermore, off-label uses for IVIG are commonly provided to patientsfor the treatment or management of various indications, for example,chronic fatigue syndrome, clostridium difficile colitis, dermatomyositisand polymyositis, Graves' ophthalmopathy, Guillain-Barré syndrome,muscular dystrophy, inclusion body myositis, Lambert-Eaton syndrome,Lupus erythematosus, multifocal motor neuropathy, multiple sclerosis(MS), myasthenia gravis, neonatal alloimmune thrombocytopenia,Parvovirus B19 infection, pemphigus, post-transfusion purpura, renaltransplant rejection, spontaneous Abortion/Miscarriage, stiff personsyndrome, opsoclonus Myoclonus, severe sepsis and septic shock incritically ill adults, toxic epidermal necrolysis, chronic lymphocyticleukemia, multiple myeloma, X-linked agammaglobulinemia, andhypogammaglobulinemia. In certain embodiments, the IVIG compositionsprovided herein are useful for the treatment or management of thesediseases and conditions.

Finally, experimental use of IVIG for the treatment or management ofdiseases including primary immune deficiency, RRMS, Alzheimer's disease,and Parkinson's disease has been proposed (U.S. Patent ApplicationPublication No. U.S. 2009/0148463, which is herein incorporated byreference in its entirety for all purposes). In certain embodiments, theIVIG compositions provided herein are useful for the treatment ormanagement of primary immune deficiency, RRMS, Alzheimer's disease, orParkinson's disease. In certain embodiments comprising dailyadministration, an effective amount to be administered to the subjectcan be determined by a physician with consideration of individualdifferences in age, weight, disease severity, route of administration(e.g., intravenous v. subcutaneous) and response to the therapy. Incertain embodiments, an immunoglobulin preparation of this invention canbe administered to a subject at about 5 mg/kilogram to about 2000mg/kilogram each day. In additional embodiments, the immunoglobulinpreparation can be administered in amounts of at least about 10mg/kilogram, at last 15 mg/kilogram, at least 20 mg/kilogram, at least25 mg/kilogram, at least 30 mg/kilogram, or at least 50 mg/kilogram. Inadditional embodiments, the immunoglobulin preparation can beadministered to a subject at doses up to about 100 mg/kilogram, to about150 mg/kilogram, to about 200 mg/kilogram, to about 250 mg/kilogram, toabout 300 mg/kilogram, to about 400 mg/kilogram each day. In otherembodiments, the doses of the immunoglobulin preparation can be greateror less. Further, the immunoglobulin preparations can be administered inone or more doses per day. Clinicians familiar with the diseases treatedby IgG preparations can determine the appropriate dose for a patientaccording to criteria known in the art.

One commonly used IgG product, intravenous immunoglobulin or IVIG, isformulated for intravenous administration. Because the IgG preparationof this invention has achieved an exceptionally high immunoglobulinconcentration (20% w/v or higher), which significantly reduces thevolume for a therapeutically effective dose, the composition of thepresent invention are particularly advantageous for subcutaneous and/orintramuscular administration to a patient, even though intravenousadministration remains an option for administration.

The term “effective amount” refers to an amount of an immunoglobulin,particularly IgG, preparation that results in an improvement orremediation of a medical condition being treated in the subject (e.g.,primary immune deficiency, RRMS, Alzheimer's disease, Parkinson'sdisease, etc.). An effective amount to be administered to the subjectcan be determined by a physician with consideration of individualdifferences in age, weight, disease severity, route of administration(e.g., intravenous v. subcutaneous) and response to the therapy. Incertain embodiments, an immunoglobulin preparation of this invention canbe administered to a subject at about 5 mg/kilogram to about 2000mg/kilogram each day. In additional embodiments, the immunoglobulinpreparation can be administered in amounts of at least about 10mg/kilogram, at last 15 mg/kilogram, at least 20 mg/kilogram, at least25 mg/kilogram, at least 30 mg/kilogram, or at least 50 mg/kilogram. Inadditional embodiments, the immunoglobulin preparation can beadministered to a subject at doses up to about 100 mg/kilogram, to about150 mg/kilogram, to about 200 mg/kilogram, to about 250 mg/kilogram, toabout 300 mg/kilogram, to about 400 mg/kilogram each day. In otherembodiments, the doses of the immunoglobulin preparation can be greateror less. Further, the immunoglobulin preparations can be administered inone or more doses per day. Clinicians familiar with the diseases treatedby IgG preparations can determine the appropriate dose for a patientaccording to criteria known in the art.

In certain embodiments, a concentrated IgG preparation can beadministered to a subject at dose of about 5 mg/kilogram to about 2000mg/kilogram per administration. In certain embodiments, the dose may beat least about 5 mg/kg, or at least about 10 mg/kg, or at least about 20mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg,300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600mg/kg, 650 mg/kg, 700 mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 mg/kg,950 mg/kg, 1000 mg/kg, 1100 mg/kg, 1200 mg/kg, 1300 mg/kg, 1400 mg/kg,1500 mg/kg, 1600 mg/kg, 1700 mg/kg, 1800 mg/kg, 1900 mg/kg, or at leastabout 2000 mg/kg.

In accordance with the present invention, the time needed to complete acourse of the treatment can be determined by a physician and may rangefrom as short as one day to more than a month. In certain embodiments, acourse of treatment can be from 1 to 6 months.

The dosage and frequency of concentrated IgG treatment will depend upon,among other factors. the disease or condition being treated and theseverity of the disease or condition in the patient. Generally, forprimary immune dysfunction a dose of between about 100 mg/kg and about400 mg/kg body weight will be administered about every 3 to 4 weeks. Forneurological and autoimmune diseases, up to 2 g/kg body weight isimplemented for three to six months over a five day course once a month.This is generally supplemented with maintenance therapy comprising theadministration of between about 100 mg/kg and about 400 mg/kg bodyweight about once every 3 to 4 weeks. Generally, a patient will receivea dose or treatment about once every 14 to 35 days, or about every 21 to28 days. The frequency of treatment will depend upon, among otherfactors. the disease or condition being treated and the severity of thedisease or condition in the patient.

In a preferred embodiment, a method of treating an immunodeficiency,autoimmune disease, or acute infection in a human in need thereof isprovided, the method comprising administering a pharmaceutical IgGcomposition of the present invention.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially the same or similar results.

Example 1 Stability Study of Various IgG Concentrations and Formulations

1. Purpose

The purpose of this study is to compare the storage stability of the lowpH (0.25M glycine pH 4.4-4.9 as measured in concentrated solution) athigher protein concentration with the neutral pH formulation (22.5 g/lglycine, 3 g/l NaCl pH 7) as currently used for intramuscularly andsubcutaneously injectable immunglobulins.

2. Experimental Design

All runs start with the concentration of the nanofiltrate to 5% protein.A ten times buffer exchange against 0.15M glycine (lowest glycineconcentration investigated) is performed followed by the finalconcentration to a target value above 20% protein. While for the firstset of experiments a 0.5 m² polyethersulfone Millipore membrane withmolecular cut-off of 30K (regular screen) is used, the second set ofexperiments is done with a 0.5 m² polyethersulfone Millipore membranewith open screen, which is more suitable for solutions with higherviscosity and the post-wash fractions are concentrated by a secondultra-/diafiltration device with a lower membrane surface (0.1 m², openscreen) in order to reduce yield losses.

The final containers are either formulated and stored at low pH or thelow pH storage is done in bulk and afterwards they are formulated atneutral pH prior to storage.

TABLE 1 Overview of the ultra-/diafiltration steps (30K polyethersulfonemembranes) and the final container pH 0.5 m² ultrafiltration devicestandard standard open open screen screen screen screen 0.1 m²ultrafiltration device none none open open screen screen Final containerpH 4.7 7 4.7 73. Test Methods

pH: pH was tested with Knick Portamess type 911 XPH equipped with aMettler Toledo LoT405-DPA-SC-S8/120 electrode and a PT 1000 temperatureprobe.

Protein: Protein values were determined using Biuret.

Molecular size distribution was tested by using high performance sizeexclusion chromatography.

ACA titer was tested as described in the European Pharmacopoeia.

4. Acceptance Criteria

The following acceptance criteria are defined:

-   -   Molecular size distribution by HPLC:        -   i. Monomers and Oligo-/Dimers: ≧90%        -   ii. Aggregates: ≦10% (≦% for IV administration)    -   ACA titer:        -   Less than 50% CH50 Units consumed/mg protein for IV            administration.            5. Results and Discussion

5.1. Comparison of Aggregate and Fragment Content and ACA Titer in thePreparations Formulated at Low (pH 4.7 and at pH 7.0)

In the following Table 2, aggregate and fragment content as well as ACAtiter after 3 months storage at 28 to 30° C. for the standardformulations (pH 4.7, 0.25M glycine; or pH 7.0, 22.5 g/L glycine, 3 g/LNaCl) at different protein concentrations are shown.

TABLE 2 Fragment, Aggregate and ACA values after 3 months storage at 28to 30° C. at low pH and pH 7.0 at different protein concentrationsFragments % Aggregates % ACA titer % Protein pH 4.7 pH 7.0 pH 4.7 pH 7.0pH 4.7 pH 7.0 14% 1.35 1.50 0.10 0.92 44.1 52.0 16% 1.24 1.38 0.08 0.9140.5 53.1 18% 1.24 1.60 0.11 0.93 40.3 52.4 20% 1.35 1.52 0.12 0.93 37.562.7

The data clearly show that the low pH formulation has lower aggregatesand lower ACA-titer after 3 months storage at 28 to 30° C. All ACAtiters of the pH 7 formulations are above the acceptance criteriondefined for this test. In Table 2 the values after 3 months storage at 2to 8° C. are given.

TABLE 3 Fragment, Aggregate and ACA values after 3 months storage at 2to 8° C. at low pH and pH 7.0 at different protein concentrationsFragments % Aggregates % ACA titer Protein pH 4.7 pH 7.0 pH 4.7 pH 7.0pH 4.7 pH 7.0 14% 0.36 1.80 0.16 1.09 38.3 46.5 16% 0.30 0.51 0.11 1.0137.4 44.7 18% 0.33 1.10 0.17 0.86 35.8 39.8 20% 0.33 1.98 0.20 1.06 36.146.0

The results at 2 to 8° C. confirm the trend seen at 28 to 30° C. The ACAtiters are all below the limit defined as acceptance criteria althoughthe pH 7.0 formulations seem to have higher values. The protein valuedoes not influence the results of the parameters tested.

5.2 Influence of Different Concentration Procedures on Aggregate andFragment Content as Well as ACA Titer in the Preparations IGSC60 (pH4.7, A-Screen) and IGSC62 (pH 4.7, V-Screen, Post-Wash Concentrationwith Smaller UF-System)

In the following Table 4, aggregate and fragment content as well as ACAtiter after 3 months storage at 28 to 30° C. for the low Ph formulationswith different concentration procedures at different proteinconcentrations are shown.

TABLE 4 Fragment, Aggregate and ACA values after 3 months storage at 28to 30° C. at low pH with different protein concentration methodsFragments (%) Aggregates (%) ACA titer standard- open- standard- open-standard- open- Protein screen screen screen screen screen screen 14%1.35 0.92 0.10 0.21 44.1 42.6 16% 1.24 1.09 0.08 0.20 40.5 40.9 18% 1.240.96 0.11 0.23 40.3 40.7 20% 1.35 0.98 0.12 0.30 37.5 41.6

The data showed similar results after 3 months storage for bothconcentration modes. In Table 5 the corresponding values at 2 to 8° C.are shown

TABLE 5 Fragment, Aggregate and ACA values after 3 months storage at 2to 8° C. at low Ph with different protein concentration methodsFragments (%) Aggregates (%) ACA titer (%) standard- open- standard-open- standard- open- Protein screen screen screen screen screen screen14% 0.36 0.27 0.16 0.17 38.3 39.6 16% 0.30 0.22 0.11 0.14 37.4 38.3 18%0.33 0.23 0.17 0.18 35.8 39.6 20% 0.33 0.22 0.20 0.20 36.1 39.9

The values obtained at 2 to 8° C. confirmed the results obtained at 28to 30° C. The concentration method does not influence the stability ofthe product.

As the approach with two systems, one for the main concentration processand the other for the concentration of the post-wash, results in higheryield, this method was judged to be more appropriate for large scalemanufacturing.

6. Conclusion

The following conclusions can be drawn from the results presented inthis study:

-   -   The low pH formulation gives lower ACA values, lower aggregate        and lower fragment contents after 3 months storage at 2 to 8° C.        and 28 to 30° C.    -   After 3 months storage at 28 to 30° C. the ACA values of the        neutral pH formulations are above the acceptance criteria.    -   The protein value does not influence the results of the        parameters tested.    -   The concentration method does not influence the stability of the        product.    -   Adequate post-wash can only be obtained with open-screen        membranes

Based on these conclusions it was decided to produce the new IgG productfor subcutaneous administration with the low pH formulation, theconcentration method using a second smaller device for concentrating thepost-wash, an ultra-/diafiltration device with open screen membranes andat a protein content of 20%±2%.

Example 2 Ultrafiltration and Formulation of SUB Q NG

1. Background

This information was gathered during production of scale-up andpre-clinical 20% lots.

The process used for manufacturing of 20% lots until the nanofiltratestep was as described above. Ultra-/diafiltration was improved toconcentrate the solution to 20% (limits: 18 to 22%). In order to reduceyield loss to a minimum, the post-wash of the ultrafiltration deviceused for diafiltration is concentrated by a second smaller deviceequipped with the same membranes and afterwards added to the bulksolution.

Surprisingly it could be shown that the virus inactivation during low pHstorage is not influenced by the protein concentration of the solution.Similar virus reduction was achieved in 10% solution (GAMMAGARD® LIQUID)and in 20% solution. Therefore low pH storage as a virus reduction stepwas maintained for the 20% product.

2. Process Narrative

Ultrafiltration

The glycine concentration of the nanofiltrate is adjusted to a target of0.25M. The solution is concentrated to a protein concentration of 6±2%w/v through ultrafiltration (UF). Typically, protein concentration isdetermined by measurement of AU₂₈₀₋₃₂₀. An extinction coefficient of14.1 is used. The pH is adjusted to 5.2±0.2. The UF membrane used has aNominal Molecular Weight Cut Off (NMWCO) of 50,000 daltons or less andis especially designed for high viscosity products (e.g., V screen fromMillipore). For example, Millipore Pellicon Biomax with a NMWCO of 50Kdaltons or less. Membrane material is polyethersulphone.

The concentrate is diafiltered against a 0.25M glycine solution, pH4.2±0.2. The minimum exchange volume is 10× of original concentratevolumes. Throughout the ultrafiltration/diafiltration operation, thesolution is maintained at 4° C. to 20° C.

After diafiltration, the solution is concentrated to a proteinconcentration of minimum 22% w/v. The solution temperature is adjustedto 2° C. to 8° C. The protein concentration may be determined by UVreading through the use of an extinction coefficient value of 14.1

In order to recover the complete residual protein in the system, thepost-wash of the first bigger ultrafiltration system is done with atleast 2 times the dead volume in re-circulation mode to assure that allprotein is washed out. Then the post-wash of the first ultrafiltrationsystem is concentrated to a protein concentration of at least 22% w/vwith a second ultra-/diafiltration system equipped with the same type ofmembrane which is dimensioned a tenth or less of the first one. Thepost-wash concentrate is added to the bulk solution. The secondultrafiltration system is then post-washed. This post-wash is used foradjustment of the protein concentration of the final bulk in step 14.The solution temperature is adjusted to 2° C. to 8° C.

Formulation

The protein concentration is further adjusted to 20.4±0.4% w/v withpost-wash of the second smaller ultrafiltration system and/or withdiafiltration buffer. The pH is adjusted to 4.4 to 4.9, if necessary.

Example 3 Manufacturing of 20% Lots

1. Introduction

This report describes the pre-clinical production and summarizes theresults of Baxter's new investigational Immunoglobulin preparation “SUBQNG, 20%,” which is a 20% (w/v) liquid polyvalent human Immunoglobulinpreparation for subcutaneous use.

The manufacturing was done as described in “Detailed Description of theInvention” with the concentration method as described above.Fractionation starts with the separation of cryo-precipitate. Thecryo-poor plasma may then be used for isolation of various crudecoagulation factors and inhibitors prior to subsequent cold alcoholfractionation. Seven pathways are chosen for batch adsorption of crudecoagulation factors and inhibitors from the cryo-poor plasma prior toSUBQ NG, 20% purification and are referred to as pathways 1 to 7 in thefollowing table.

TABLE 6 Absorption Pathways Step Gel Heparin 1 2 3 4 5 6 7 Cryo- — — X XX X X X X precipitation FEIBA 0.5 g DEAE- — X X Sephadex/1 Factor IX 0.5g DEAE-  2000 X X X X Sephadex/1 IU/ml Factor VII 120 mg  750 X XAl(OH)₃/1 IU/ml Antithrombin 1 g DEAE- 80000 X X X Sephadex/1 IU/ml

For pre-clinical SUBQ NG, 20% production Cohn starting materials derivedfrom the pathways 1, 3 and 6 were chosen to cover a broad variety ofdifferent adsorption options. Various adsorption processes are describedin Teschner et al., 2007, Vox Sang. 92:42-55; Polsler et al., 2008, VoxSang. 94:184-192; U.S. Pat. Nos. 6,395,880 and 5,409,990; andProthrombin complex: Brummelhuis in Methods of Plasma ProteinFractionation (J. M. Curling Editor, Academic Press, 1980).

The modified Cohn alcohol fractionation leads to a principalintermediate IgG fraction, referred to as Precipitate G, which isfurther processed to the final product using chromatographicpurification. The downstream manufacturing comprises cation exchange(CM-Sepharose fast flow) and anion exchange chromatography(ANX-Sepharose fast flow) and includes three independent virusinactivation or removal steps, namely

-   -   Solvent/detergent treatment (mixture of 1% Triton X-100, 0.3%        Tri-N-butyl phosphate and 0.3% Polysorbate-80),    -   Nanofiltration (Asahi Planova 35 nm) and    -   Low pH storage for 3 weeks at elevated temperature.

In order to reach a higher protein concentration for subcutaneousapplication an open channel screen has to be used at theultra-/diafiltration step. Preferably a second ultra-/diafiltrationdevice is used for the concentration of the post-wash fraction in orderto recover all protein from the first device.

SUBQ NG, 20% is a liquid formulation of Immunoglobulin G (IgG), of whichat least 95% of the protein is gamma globulin. The product is isotonicand formulated at low pH. At a concentration of 10%, protein during thefinal concentration step the pH is 4.4 to 4.9. The final pH of the 20%solution will be determined after the results of extended stabilitystudies are available. The final solution contains 180 to 220 g proteinand as the only excipient 0.1 to 0.3 moles of Glycine per litersolution. The liquid preparation is clear to slightly pale-yellow andsubstantially free of visible particles.

2. Data and Mass Balance of the Pre-Clinical Lots

-   -   1. Pre-Clinical Lot: SC00107NG        -   Adsorption Option 1: Option 6: F IX, F VII, AT-III        -   Lot number of starting material: Precipitate G VNELG171            (US-source)        -   Lot number of final container: SC00107NG    -   2. Pre-Clinical Lot: SC00207NG        -   Adsorption Option 3: FEIBA, AT-III        -   Lot number of starting material: Precipitate G VNELG 173            (US-source)        -   Lot number of final container: SC00207NG    -   3. Pre-Clinical Lot: SC00307NG        -   Adsorption Option 1: no adsorption steps        -   Lot number of starting material: Precipitate G LB0790301            (US-source)        -   Lot number of final container: SC00307NG

TABLE 7 Step Sterile Bulk Test/Method Lot SC00107NG SC00207NG SC00307NGTotal g/L Plasma 3.4 3.7 3.7 protein/UV IgG/Nephel- g/L Plasma 3.0 3.03.0 ometric IgA/ELISA g/L Plasma <0.001 <0.001 <0.001 IgM/ELISA g/LPlasma <0.001 <0.001 <0.001 MSD % Aggregates 0.1 0.1 0.1 (HPLC) % Oligo/4.6 4.5 3.2 Dimers % Monomers 95.2 95.4 96.6 % Fragments 0.1 0.1

At the final bulk level the purity of the preparation is illustrated bythe low levels of the main impurities, which are well below 0.1% of thetotal IgG. The molecular size distribution in the 20% product at thisfinal stage of the process is similar to the one of aGAMMAGARD®LIQUID/KIOVIG final container. This indicates that theconcentration to 20% protein has no negative impact on the integrity ofthe IgG molecule.

3. Additional Results from the Characterization of the Pre-ClinicalBatches

The preliminary final container release criteria were defined on thebasis of the requirements from the authorities for subcutaneous humanimmunoglobulins, the final container specifications of the currentproduct for subcutaneous administration and the GAMMAGARD®LIQUID/KIOVIGspecifications. Additional Quality Control tests were performed toevaluate the level of product and/or process related impurities.

Furthermore the characterization of the relevant Antibody Spectrum ofthe Final Containers was done and compared to the results from thepre-clinical IVIG, 10% TVR lots.

The results are given in the following table (Table 8)

IVIG, 10% TVR SUBQ NG 20% P0010ING P00201NG P0030ING Test System UnitSC00107NG SC00207NG SC00307NG 01C21AN11 0IC21AN21 01D05AN11 Bacteria:Corynebacterium Guinea pigs IU/ml 6. 5. 5. 5. diphtheriae EUR VirusesHAV ELISA IU/ml 14. 14 1 HBV (antibody to ELISA IU/mg 40. 35.9 40.1 40.hep B s Ag) TP Measles virus Hemagglut. 41. n.a. n.a. n.a EUR Enrich.Factor Measles virus US Hemagglut. NIH 176 0.8 1.001 1.0 1.001 Parvo 619ELISA IU/ml 718 78 71 567 442 36 Poliomyelitis NIHU/ 1.4 1.711 1.5 1.011.11 1.21 virus type I ml

The antibody titers and the enrichment factors are in the same order ofmagnitude for the three pre-clinical SUBQ NG 20% final containers andfor the pre-clinical GAMMAGARD®LIQUID/KIOVIG lots.

Quality Control Tests of SUBQ NG 20% Final Container (Table 9)

Test System Unit SC00107NG SC00207NG SC00307NG Fc functional integrityBc-binding % of BPR lot 3 15.8 138 164 Anti-complementary activity EPmethod % 41.1 41.5 41.2 Anti-complementary activity EP method C′H50 U/mg41.4 41.8 41.6 Prekallikrein activator activity, EUR chromogenic IU/ml<0.6 1.004 1.237 Anti-A hemagglutinins, pH. Eur. hemagglut. Dilution: 1:8 16 8 Anti-B hemagglutinins, pH. Eur. hemagglut. Dilution: 1 4 4 2Anti-D hemagglut. complies complies Complies Exclusion of pyrogenicity,pH. Eur. rabbit ° C. rise pyrogen free pyrogen free pyrogen free and CFRBacterial Endotoxins Chromogenic IU/ml <1.2 1.8 <1.2 Purity by celluloseacetate CAE % 99.6 99.8 99.5 electrophoresis Molecular size distributionSE-HPLC % 99.2 99.3 99.2 (Monomer + Dimers) Molecular size distributionSE-HPLC % 0.2 0.2 0.3 (Polymers) Molecular size distribution SE-HPLC %0.6 0.5 0.5 (Fragments) IgA-EUR ELISA μg/ml 20 20 30 IgM ELISA μg/ml 1.11.0 1.2 IgG Nephelomnetry mg/ml 177 165 163 Protein (Bulk) UV mg/ml 201203 202 Protein Autom.N2 mg/ml 202 208 203 Glycine HPLC mg/ml 14.7 14.514.7 Polysorbate 80 Spectrophot. μg/ml <250 <250 <250 TNBP Gas-chromat.μg/ml <0.3 <0.3 <0.3 Octoxynol 9 Ion-chromat. μg/ml <3 <3 <3 SterilityMembrane filtr. NA sterile sterile sterile Osmolality mOsmol/Kg 298 298299 pH, undiluted Potentiometry 5.1 5.2 5.3 Appearance Visual Inspec.satisfied satisfied satisfied Ethanol Gas-chromat. μg/ml <20 <20 <20Isopropanol Gas-chromat. μg/ml <20 <20 <20 Aluminum AAS Photometry μg/L<50 <50 <50 Silicium ICP OES Ion Electr. μg/L 3466 17270 21180 HeparinIU/ml <0.0075 <0.0075 <0.0075

The removal of product and process related impurities is satisfactoryfor all three lots.

4. Conclusions

Three final container lots of SUBQ NG 20% were successfully manufacturedin the 200 liter scale. Three adsorption pathways were chosen to cover abroad variety of adsorption steps prior to alcohol fractionation,namely:

Option 1, US source plasma without adsorption steps

Option 3, US source plasma after FEIBA, AT-III adsorption

Option 6, US source plasma after F-IX, F-VII, AT-III adsorption

In process parameters monitored during the pre-clinical production andthe characterization of intermediates and the final product showed thatthere are no obvious differences detectable between the three lots.

All final containers meet the product related preliminary specificationsregardless which kind of starting material was chosen.

Example 4 Storage Study of 20% Preparation

1. Introduction

SUBQ NG, 20% is a 20% (w/v) liquid polyvalent human Immunoglobulinpreparation for subcutaneous use. The SUBQ NG, 20% was produced asdescribed above.

This study summarizes the storage data of 3 preclinical lots and onefeasibility lot at 2 to 8° C. and 28 to 30° C. (feasibility lot only)for up to 6 months.

2. Purpose

The purpose of this study is to evaluate the storage stability of Sub QNG 20% final containers at 2 to 8° C. and 28 to 30° C.

3. Stability Indicating Parameters

The primary degradation modes are denaturation, aggregation andfragmentation, resulting in a change in the molecular size distributionof the sample. Therefore the molecular size distribution analysis byHigh Performance Size Exclusion Chromatography is the main stabilityindicating parameter.

4. Batches Examined and Primary Packaging

The stability data presented in this report are made with thepre-clinical lots SC00107NG, SC00207NG and SC00307NG as well as with thefeasibility lot IgGSC 62/1.

5. Results

In the following Table 11 the results of the pre-clinical finalcontainers after storage up to 12 months are shown.

TABLE 10 Stability of pre-clinical Sub Q 20% batches at 2 to 8° C. MSD(HP-SEC) (%) Aggregates Olig/Dimers + Fragments Lot Month (>450 KDa)Monomers (<70 Kda) SC00107NG 0 0.3 99.5 0.2 3 0.4 99.5 0.2 4 0.5 99.40.2 6 0.5 99.3 0.2 12 0.7 99.1 0.3 SC00207NG 0 0.3 99.5 0.2 3 0.4 99.50.1 4 0.5 99.3 0.2 6 0.6 99.2 0.2 12 0.8 99.0 0.2 SC00307NG 0 0.3 99.60.1 3 0.5 99.3 0.2 4 0.6 99.2 0.1 6 0.7 99.1 0.2 12 0.9 98.8 0.2 Releasecriteria <5 >90 <5

TABLE 11 Stability of the feasibility lot IgGSC 62/1 at 2 to 8° C. and28 to 30° C. MSD (HP-SEC) (%) Aggregates Olig/Dimers + Fragments Lot °C. Month (>450 KDa) Monomers (<70 Kda) IgGSC 2 to 8 0 0.2 99.5 0.3 62/11 0.1 99.7 0.2 3 0.2 99.6 0.2 6 0.3 99.4 0.3 12 0.4 99.3 0.3 18 0.4 99.20.4 28 to 30 0 0.2 99.5 0.3 1 0.2 99.2 0.6 3 0.3 98.7 1.0 6 0.6 98.0 1.412 1.2 95.6 3.2 18 1.9 93.5 3.8 Release <5 >90 <5 criteria6. Conclusion

The data confirmed that the product complies to the pre-definedspecifications for the parameters investigated for up to 18 monthsstorage at 2 to 8° C. and 28 to 30° C.

Example 5 Low DH Treatment for Viral Inactivation

Purpose and Rationale

Introduction

Immune Globulin Subcutaneous (Human), 14%-20%, Triple Virally Reduced(TVR) Solution, in the following called Subcuvia NG Solution, ismanufactured from pooled human plasma. After mass capture steps toremove coagulation factors/inhibitors, purification of Subcuvia NGstarts with a modified Cohn alcohol fractionation, leading to aprincipal intermediate precipitate G, followed by a downstream processwhich contains chromatographic purification as well as three distinctvirus inactivation/removal steps. In the current study, the virusreduction capacity of the low pH storage of the final container wasinvestigated in detail.

The downstream purification process comprises the following steps:resuspended Precipitate G is subjected to Solvent/Detergent (S/D)treatment, cation exchange chromatography usingCarboxymethyl(CM)-Sepharose fast flow and anion exchange chromatographyusing ANX-Sepharose® fast flow, nanofiltration, ultra-diafiltration, pHadjustment, and sterile filtration and filling. After aseptic fillingthe Subcuvia NG Solution is subjected to low pH storage in the finalcontainer (for schematic illustration of the manufacturing process, seeprocess scheme below).

Process Scheme

Process scheme A, shown in FIG. 2, provides an overview of thedownstream part of the manufacturing scheme for Subcuvia NG Solutionstarting with Step 10. The custom-made intermediate used in the currentstudy is equal to the process step “Final container, Pre-storage.”

Process scheme B, shown in FIG. 2, provides an overview of thedownscaled process used in the present study including sample drawingfor virus titration.

Purpose

To provide a high safety margin with respect to a potential virustransmission, three dedicated virus inactivation/removal steps, whichcomplement each other in their mode of action, are integrated into themanufacturing process of Subcuvia NG Solution:

-   -   Solvent/Detergent treatment (step 11)    -   Nanofiltration (step 14)    -   Low pH Storage at elevated temperature in the final container        (post-aseptic filling)

The capacity and robustness of the storage at low pH and elevatedtemperature with respect to inactivation of viruses was alreadyinvestigated in the course of a previous study, in which IGIV 10% TVR, aproduct equivalent to Subcuvia NG but adjusted to 10% protein for i.v.application, was used. The results obtained from this study demonstratedthat all lipid-enveloped viruses investigated were effectively androbustly inactivated, with Bovine viral diarrhea virus (BVDV) showingthe slowest inactivation kinetics. Moreover, it could be demonstratedthat this process step further contributes to the viral safety of themanufacturing process with regard to small non lipid-enveloped DNAviruses. Subcuvia NG and IGIV 10% TVR are immunoglobulin products, whereSubcuvia NG is the variation for subcutaneous administration, and IGIV10% TVR is the product variation for i.v. application. Both share thesame manufacturing process up to the ultrafiltration/diafiltration andformulation steps, where the only difference is that the proteinconcentration of Subcuvia NG is adjusted to a range of 14% to 20%instead of 10%.

Therefore the capacity and robustness of the storage step at low pH andelevated temperature in the manufacture of Subcuvia NG (see, TheEuropean Agency for the Evaluation of Medicinal Products Evaluation ofMedicines for Human Use (2001): CPMP Biotechnology Working Party—Notefor Guidance on Plasma-Derived Medicinal Products, CPMP/BWP/269/95 (rev.3)) with respect to inactivation of BVDV and MMV was evaluated bysetting selected critical process parameters to the upper and lowerlimits specified for the manufacturing process (i.e., time, temperature:lower limit; pH: upper limit). In addition, to further investigate therobustness of this virus inactivation step, the temperature was reducedfor a given time period during the downscaled runs.

As discussed above, the following enveloped and non-enveloped viruseswere used.

-   -   Bovine viral diarrhea virus (BVDV) as a model for hepatitis C        virus (HCV) and for other small lipid-enveloped RNA viruses.    -   Mice Minute Virus (MMV) as a model for Human Parvovirus B19        (B19V) and for other small non-enveloped DNA viruses.

In accordance with the CPMP guideline 268/95 (The European Agency forthe Evaluation of Medicinal Products Human Medicines Evaluation Unit(1996): CPMP Biotechnology Working Party—Note for Guidance on VirusValidation Studies: The Design, Contribution and Interpretation ofStudies Validating the Inactivation and Removal of Viruses,CPMP/BWP/268/95 (revised)) the study was conducted with a downscaledmodel of the respective manufacturing step. The validity of the resultsobtained with the scaled-down manufacturing step “Storage at low pH andelevated temperature” with regard to virus inactivation in themanufacture of Subcuvia NG Solution, was demonstrated by comparison ofparameters specified for this manufacturing step in the large-scaleproduction process and process parameters of the downscaled model.Additionally, selected biochemical parameters were monitored andcompared to the respective parameters of the large-scale process.

Materials, Methods and Equipment

Viruses and Cells

BVDV, strain NADL (biologically cloned, ATCC VR-1422), obtained from theAmerican Type Culture Collection (ATCC; Rockville, Md.) is used. Thevirus is propagated on MDBK cells (ATCC CCL-22), according to standardoperating procedures, and titrated on BT cells (ATCC CRL-1390).

MMV, prototype strain (ATCC VR-1346), was obtained from the AmericanType Culture Collection, Rockville, Md. The virus was propagatedaccording to standard operating procedures and titrated on A9 cells(ATCC CCL-1.4).

Test Item

The following lots of custom-made Subcuvia NG intermediate, before“Storage at low pH and elevated temperature”, i.e., after manufacturingstep aseptic filling, were obtained from Baxter's PPD Product Supportdepartment, Industriestrasse 131, Vienna, Austria:

-   -   Lot number IGSC64, with a protein concentration of 13.5%    -   Lot number IGSC64, with a protein concentration of 20.9%

The material was shipped in the final container at +2° C. to +8° C. andwas used within 6 months.

Buffers/Solutions

Solutions for pH Adjustment

The pH was adjusted using 0.5 M NaOH or 0.5 M HCl. (Both solutions werestored at room temperature and used within 12 months).

Preparation of 0.5 M Hydrochloric Acid (HCl)

Component Component per liter solution Shelf life Storage HCl 37% 41.4 ±0.4 ml 12 months 23 ± 5° C.

Reagents, i.e. aqua dest. and HCl were combined at ambient temperature.

Preparation of 0.5 M Sodium Hydroxide (NaOH)

Component Component per kg of aqua dest. Shelf life Storage NaOH 20.0 ±0.2 g 12 months ±5° C.

The respective amount of NaOH was dissolved in aqua dest. at ambienttemperature.

Solutions for pH Measurement

pH was measured both directly and in a diluted solution according to theEuropean Pharmacopoeia (EP). For measuring pH according to the EPmethod, the protein concentration was diluted to 1% using a 0.9% NaClsolution.

The 0.9% NaCl solution was prepared as follows: 9.0±0.9 g NaCl weredissolved in 1000 ml aqua dest. The solution was 0.2 μm filtered, storedat room temperature and used within 12 months.

Cell Culture Media

Media used for cell culture or virus titration were prepared accordingto standard operating procedures for BVDV and MMV viral titration.

Virus Titration Assays

TCID₅₀ Assay

Samples containing viruses were titrated by TCID₅₀ assays according tostandard operating procedures. Briefly, serial ½ log dilutions of thesamples were prepared in the respective tissue culture medium, and 100μl of each dilution were added to each of 8 wells of a microtiter plateseeded with the respective indicator cell line. The microtiter platesare stored in humidified and CO₂-regulated storage units at atemperature of 36° C.±2° C. Evaluation of cytopathic effects isaccomplished by visual inspection of the cells under a microscope after7 days of storage.

Median tissue culture infectious doses (TCID₅₀) were calculatedaccording to the Poisson distribution and expressed as log₁₀[TCID₅₀/ml].

BVDV Bulk Titration

Studies were performed on MMV-spiked samples. In order to lower thelimit of detection for the BVDV spiked samples taken from the SubcuviaNG intermediate after 20 and 27 days of low pH storage, these sampleswere titrated as follows: in addition to the standard TCID₅₀ assay,samples of the 1:3.16 dilution (0.5 logs) and following two ½ logdilutions (the respective cell culture medium is used for dilution) areadded to all wells (100 μl per well) of 96-well microtiter plates seededwith the respective indicator cell line. Storage of cells and evaluationof the cytopathic effect of the respective virus are done as describedabove (TCID₅₀ Assay). The results were calculated as follows: The ratioR_(Vol) of the volume titrated in the bulk titration and the volumetitrated in the regular TCID₅₀ assay was calculated. The log₁₀ of RVolis subtracted from the virus titer determined in the TCID₅₀ assay, andthe result is given as the virus titer determined by bulk titration.

Example

-   -   A sample is assayed (TCID₅₀ assay) on a microtiter plate in        serial 0.5 log dilutions (all 12 columns) and the wells are        found negative from the dilution 0 (i.e. undiluted) onwards.        Calculation according to the Poisson distribution gives a virus        titer of <0.11 log₁₀ [TCID₅₀/ml]. The corresponding total volume        of the sample assayed is 1.17 ml.    -   The same sample (dilution 0) is applied onto all wells of a        microtiter plate (bulk titration). Therefore, the total sample        volume applied is 9.6 ml    -   The ratio of volumes is: R_(Vo)l=9.6:1.17=8.21; log₁₀        (R_(Vol))=0.91.    -   The calculated virus titer is then <0.11-0.91; i.e. <−0.80. The        upper limit of the virus titer's confidence interval is        calculated identically.        Calculation of virus titer when no infectivity is scored in        successive kinetic samples

Where no viral infectivity was detected in successive kinetic samples upuntil the final sample after completion of low pH storage, the volume ofall successive negative samples used for wells in the TCID₅₀ assay witha clear negative result as taken into account for calculation of theassay detection limit. For this the following formula was used (see alsoAppendix 1):

${{Virus}\mspace{14mu}{titer}\mspace{14mu}{based}\mspace{14mu}{on}\mspace{14mu} n\mspace{14mu}{negative}\mspace{20mu}{successive}\mspace{14mu}{kinetic}\mspace{11mu}{samples}},{\left\lbrack {\log_{10}\left( {{TGCID}_{50}/{ml}} \right)} \right\rbrack = {< {- {\log_{10}\left( {\sum\limits_{i = 1}^{n}\;{/10^{Xi}}} \right)}}}}$with

X_(i) (i=1, 2, 3, . . . n) individual virus titers [log₁₀ (TCID₅₀/ml)]of n successive negative samples

If all negative samples have the same titer, i.e. x, the formulasimplifies to:

Virus  titer  based  on  n  negative  successive  kinetic  samples, [log₁₀(TGCID₅₀/ml)] =  < x − log₁₀(n)Cytotoxicity

The cytotoxicity of process intermediates for the virus indicator celllines used was determined from mock-spiked Subcuvia NG intermediate,i.e. spiked with BT-medium for infected cells (5% Horse serum)* insteadof virus stock as well as from mock-spiked, pH adjusted (pH 4.9±0.1) and0.45 μm filtrated starting material on each cell line for determinationof cytotoxicity. Samples were assayed like virus-containing samples,i.e. they were serially diluted and Stored with the indicator cell line.After storage, the highest non-cytotoxic concentration was determined.With regard to variations in these biological systems, deviations of±0.5 log steps were considered non-significant when comparingcytotoxicity in virus spiked and mock-spiked samples.

-   -   * The composition of the BT-medium for infected cells is as        follows: DMEM (containing 4.5 g/l D-Glucose)+1% (v/v)        L-Glutamine (200 mM)+1% (v/v) Gentamicin Sulphate (10 mg/ml)+1%        (v/v) Sodium Pyruvate+2% (v/v) Sodium Bicarbonate (7.5%)+1%        (v/v) non-essential amino acids+5% (v/v) Horse serum.        Interference

For samples with low virus titers, the influence of the sample matrix onthe performance of the infectivity assay needs to be assessed. Forsamples containing high virus titers, the relevant part of the dilutionseries, i.e. the part where virus-positive and virus-negative wells canbe scored, occurs at high sample dilutions. Consequently, the influenceof the sample matrix, which is also diluted several log₁₀ steps, on thedetection of virus infectivity can be neglected. The interference withdeterminations of low viral titers for samples containing Subcuvia NGintermediate was measured as follows: Samples were drawn from themock-spiked, pH adjusted (pH 4.9±0.1) and 0.45 μm filtrated startingmaterial (Subcuvia NG intermediate), diluted 1:3.16 with the appropriatetissue culture medium and spiked 1:100 with pre-diluted (in appropriatetissue culture medium, i.e. BT medium for BT cells/BVDV and A9 mediumfor A9 cells/MMV) virus stock suspension to a nominal titer of 2 or 3log₁₀ [TCID₅₀/ml] and titrated as described above. The titers obtainedfor spiked process intermediates were compared to those of spiked cellculture medium controls.

Calculation of Virus Reduction Factors

The analysis of the virus inactivation capacity of the process wascarried out according to CPMP guideline 268/95 [3], using the followingformula:

$R = {\log\left( \frac{V_{1} \times T_{1}}{V_{2} \times T_{2}} \right)}$where,

R=virus reduction factor

V1=volume of starting material

T1=concentration of virus in starting material [TCID₅₀/ml]

V2=volume of material after the step

T2=concentration of virus after the step [TCID₅₀/ml]

The volumes and the titers of each spiked sample before and aftertreatment were used to calculate R. Whenever virus was undetectable, thedetection limit was taken as the virus titer for calculation. Accordingto standard operating procedures, calculations were carried out withvirus titers (log₁₀[TCID₅₀/ml) given to two decimal places; reductionfactors were rounded to the first decimal place.

Determination of Validation Parameters

Time, Temperature [° C.]

Storage time was measured with timers; the temperature was measured withPt-100 sensors and recorded continuously.

pH-Value

The pH value was determined according to standard operating procedureswith a standard laboratory pH meter by direct measurement as well asimplementing the method specified by the European Pharmacopoeia (EP),i.e. after dilution to 1% protein with 0.9% NaCl solution. In thefollowing, pH with suffix (EP) means pH measurement according to EPmethod, whereas “pH” without suffix means direct measurement.

Determination of Other Parameters

Molecular Size Distribution (MSD)

HPLC-SEC (High-Performance Liquid Chromatography—Size ExclusionChromatography) analysis of molecular size distribution was done by thedepartment Analytical Biochemistry, Baxter, Vienna, Austria, accordingto standard operating procedures. The HPLC-SEC analysis of all samplesof the “scale-up” lots (produced in the department PPD Product Support,Baxter, Vienna, Austria) were performed according to this test. Thus, toassure comparability between the test results of the samples of themock-spiked runs (accumulated in the downscaled process in the course ofthis study) and the test results of the “scale-up” lots, the analysis ofMSD of the samples of the mock-spiked runs were done according to thesame test in the same department.

Cellulose Acetate Electrophoresis

Cellulose Acetate Electrophoresis (CAE) was performed according tostandard operating procedures by the department ACV Chemistry, Baxter,Vienna, Austria.

Rounding

Rounding was performed according to standard practices:

-   -   Assay results were rounded to the same number of decimal places        as specified for the respective parameter in the manufacturing        process, or as determined by the accuracy of the assay.    -   Calculations were done without rounding of assay results, and        only the final result was rounded.        Equipment    -   A standard laboratory pH-meter was used.    -   A Mettler AT-100 analytical and a Sartorius BP3100P laboratory        balance were used.    -   0.45 μm filtration was performed with PVDF membrane filters        (Millex-HV, or equivalent).    -   The process time was measured with laboratory timers.    -   Biosafety class II cabinets were used.    -   Cells were stored in humidity-controlled Heraeus CO₂-incubators.    -   Magnetic stirrers/stirring bars were used for mixing.    -   Temperature was controlled by Haake or Julabo cryostats and/or a        stirring heating block.    -   Temperature was measured using Pt-100 sensors connected to a μR        1000 YOKOGAWA recorders.        Study Design

In order to investigate the robustness of manufacturing step “Storage atlow pH and elevated temperature” of Subcuvia NG with respect to virusinactivation, key parameters for the efficacy of virus inactivation weredefined as discussed below. Runs were performed under conditionssuitable to investigate the robustness of virus inactivation by low pHstorage. A comparison of the conditions for the large-scale process andthe downscaled runs is given in Table A.

-   -   Protein concentration: As Subcuvia NG is a developmental        product, the protein concentration range for the formulated bulk        is currently rather broadly defined, i.e. approximately 14% to        20%, and might be narrowed down later on. Thus, to investigate        the robustness of the low pH treatment, two runs (run designs)        were performed at the two extremes of possible protein        concentrations. Run design 1 was performed at 13.5% protein        concentration; Run design 2 was performed at 20.9% protein        concentration.    -   The protein concentration of the custom-made material was        provided by the department PPD Product Support. The protein        concentration of the custom-made material was not determined        again, as data were already available from analyses, performed        after production of the custom-made Subcuvia NG intermediate.        Since the material, which was used for the down-scaled process,        was sterile filtered and stored at 2° C. to 8° C. until the        start of the down-scaled process (i.e., not subjected to any        freeze/thaw procedure), no change in the protein concentration        is anticipated for the finally formulated and sterile filtered        intermediate.    -   pH-Value: In production, a pH range of 4.4-4.9 (direct        measurement) is specified for the Subcuvia NG final container        throughout the low pH storage. To investigate the efficacy and        robustness of the low pH treatment under conditions less        favorable for virus inactivation both runs were performed at the        higher limit specified, i.e., a pH of 4.9±0.1. The pH was        measured after spiking and adjusted to the higher limit, if        necessary. After the low pH storage for 27 days±5 hours, the pH        was measured again by direct pH-measurement and by the method        recommended by the European Pharmacopoeia, i.e., dilution to 1%        protein with 0.9% NaCl solution.    -   Temperature: In the production process the temperature during        low pH storage is set to 30-32° C. To investigate the robustness        of the low pH storage under conditions less favorable for virus        inactivation, all downscaled runs were performed at the lower        limit of the temperature range, i.e., at 30±1° C. To examine the        potential impact of temperature fluctuations, the temperature        was reduced to 25±1° C. for ≧6 hours once every week (i.e.,        decrease in temperature was started during day 0, 7, 14, 20 and        day 27 of storage; numbering of days refers to calendar days,        i.e. day 0 is the day on which the storage is started).        Erroneously, the decrease in temperature to 25±1° C. for 6 hours        was performed twice per week in the first and in the second week        of storage, i.e., during day 0 and 4 of storage in the first        week and during day 7 and 10 of storage in the second week of        the low pH treatment.    -   Time: The storage time is typically in the range of 21 days        (required for effective inactivation of BVDV) to 28 days (upper        limit for technical/logistical reasons). To investigate the        efficacy and robustness of the low pH treatment under conditions        less favorable for virus inactivation all downscaled runs were        performed below the limits of the above listed storage time        range; i.e. for 20 days±4 hours for the short option and storage        for 27 days±5 hours for the longer option. The spiked        intermediate was stored at low pH and elevated temperature for        27 days±5 hours, but samples for virus titration were drawn        after 20 days±4 hours of storage, in order to investigate virus        clearance after 3 weeks of storage (see also Table A).

Overall, a total of 4 virus-spiked runs (2 with BVDV, and 2 with MMV)were performed to investigate the robustness of the virus inactivationcapacity of manufacturing step “Storage at low pH and elevatedtemperature.” Furthermore, two unspiked control runs were performed togenerate samples for determination of biochemical parameters. Inaddition, two mock-spiked control runs were performed and samples takenfrom these runs were used to investigate potential effects (i.e.,cytotoxicity and interference) of process intermediates on the virustitration assay.

To further demonstrate the equivalence of the downscale and the largescale processes, the following biochemical analyses were performed onthe final product before and after low pH storage: molecular sizedistribution and cellulose acetate electrophoresis.

TABLE A Process and biochemical parameters of the down scale runs,compared to the large-scale manufacturing process. Parameters, whereranges different from the large-scale process apply for the downscaledprocess, are in bold print. large-scale down-scaled process Parameterprocess Run design 1 Run design 2 Before spiking and pH adjustmentProtein concentration [g/100 ml] 14 to 20¹    14 ± 1 ¹    20 ± 1¹ Afterspiking and pH adjustment pH (direct measurement) 4.4-4.9   4.9 ± 0.1  4.9 ± 0.1 Low pH storage Storage period [d]  21-28² 20 d ± 4 h ³ 20 d± 4 h ³ 27 d ± 5 h ³ 27 d ± 5 h ³ Temperature during storage  30-32   30 ± 1 ⁴    30 ± 1 ⁴ [° C.] After low pH storage pH (directmeasurement) 4.4-4.9 as measured (not specified) pH (diluted to 1%protein with 4.6-5.1 as measured (not specified) 0.9% NaCl) EP⁵ ¹AsSubcuvia NG is a developmental product, the protein concentration is notnarrower defined yet. Thus, the two down-scaled runs were performed atthe two extremes of possible protein concentration. ²The storage periodis not narrower defined yet, as dependent on the outcome of the virusclearance studies. ³Both runs were performed for 27 d ± 5 h and samplesfor virus titration were taken on day 20 of storage so that virusinactivation data were also available for the large scale process optionof 21 to 22 days of storage. ⁴The temperature was reduced to 25 ± 1° C.for · 6 hours once every week (i.e. decrease in temperature was startedduring day 0, 7, 14, 20 and day 27; numbering of days refers to calendardays, i.e. day 0 is the day on which the storage is started) of storage.As discussed in Section 4.1.1 (“Temperature profile”), the decrease intemperature to 25 ± 1° C. for 6 hours was erroneously performed twiceper week in the first and in the second week of storage (see alsoDR1_0407). ⁵Method according to the European Pharmacopoeia.Experimental Procedure

Due to its complexity, the sequence of virus spike, pH adjustments and0.45 mm filtration steps is illustrated in the flow chart found in FIG.3 (thickness of arrows indicates relative volumes).

Starting Material

For Run 1 custom-made material (Lot number IGSC64) with a proteinconcentration of 13.5% was taken. This material was used to perform runsat the lower limit of the possible protein concentration, at the upperlimit of the pH range (i.e., BVDV: 4.97 and MMV: 4.93) and at the lowerlimit of the temperature range (i.e., at 29.4° C.).

For Run 2 custom-made material (Lot number IGSC64) with a proteinconcentration of 20.9% was taken. This material was used to perform runsat the upper limit of the possible protein concentration, at the upperlimit of the pH range (i.e., BVDV: 4.92 and MMV 4.86) and at the lowerlimit of the temperature range (i.e., 29.4° C.).

Virus Spike: BVDV, MMV

45.5 ml of each starting material was spiked with 4.5 ml of virus stocksuspension, and a 1 ml-sample was drawn after 1 to 2 minutes ofstirring. The sample was diluted immediately 1:3.16 (i.e., 1 volume ofsample plus 2.16 volumes of cell culture medium) with the respectivecell culture medium and titrated. Subsequently, another 3 ml sample wasdrawn and filtered through a 0.45 μm PVDF membrane. One ml of thefiltered material was diluted immediately 1:3.16 with the respectivecell culture medium and titrated.

Adjustment of pH

An aliquot of 35 ml of the virus spiked starting material was adjustedto a pH of 4.9±0.1 (both runs) using a 0.5 M HCl solution understirring. The material was then divided into two aliquots. One aliquotof 30 ml was used for the “Low pH Storage” and a 5 ml aliquot was usedfor the “temperature hold control” (see section “Hold Controls”).

Another aliquot of 10 ml of the virus-spiked starting material wasadjusted to a pH of 7.0±0.1 using a 0.5 M NaOH solution, where 5 ml ofthe pH-adjusted material were used for the “pH Hold Control”.

The remaining 5 ml of the pH adjusted material were used for the“Combined pH and Temperature Hold Control” (see section “HoldControls”).

Hold Controls

To investigate the mechanism of virus inactivation, i.e. by pH or bytemperature, three hold controls were stored under different conditions:

Each of the 5 ml-aliquots of the virus-spiked and pH adjusted (pH7.0±0.1) starting material were filtered through a 0.45 μm PVDF membranefilter into a cryovial. One aliquot was stored at 30±1° C., togetherwith the spiked process material and then kept at this temperature untilthe end of the process (pH Hold Control, “pH HC”). The other aliquot wasimmediately stored at +2° C. to +8° C. for 27 days±5 hours (Combined pHand Temperature Hold Control, “c HC”).

The 5 ml-aliquot of the virus-spiked, pH-adjusted (pH 4.9±0.1) and 0.45μm filtered starting material (see above “Adjustment of pH”) wasimmediately stored at +2° C. to +8° C. until the end of the process(Temperature Hold Control, “t HC”).

The minimum volume of all Hold Controls after filtration was always morethan 3 ml, thus, a proper volume for virus titration after the storageperiod was available.

Low pH Storage

The 30 ml-aliquot of the virus spiked and pH adjusted (pH 4.9±0.1)Subcuvia NG intermediate was filtered through a 0.45 μm PVDF membranefilter into a 50 ml sterile glass bottle. The minimum volume of theSubcuvia NG intermediate after filtration was always more than 24 ml.Thus, enough volume for virus titration after the storage period wasavailable. Subsequently, the temperature was equilibrated to 30±1° C.under agitating back and forth in slow motion using a water bath, withthe water bath regulated by a temperature controlled cryostat via anexternal temperature sensor. The external temperature sensor and anadditional Pt 100 electrode were placed into one 50 ml glass bottleequivalent to those used for the low pH storage, each of them filledwith 30 ml water, which was the maximal possible amount of spikedprocess material after filtration. The temperature was recordedcontinuously. As soon as the material reached a temperature of 29° C.,sample taking was initiated. Each sample was immediately diluted 1:3.16(i.e. 1 volume of sample plus 2.16 volumes of cell culture medium) withthe respective cold cell culture medium (stored at +2° C. to +8° C.) toprevent further inactivation of virus by low pH, and titrated. In allruns the Subcuvia NG intermediate was stored under agitation (back andforth in slow motion) at 30±1° C. throughout the whole process for 27days±5 hours, with a reduction of the temperature to 25±1° C. for atleast six hours (≧6 h) once every week of storage. Erroneously, thedecrease in temperature to 25±1° C. for 6 hours was performed twice perweek in the first and in the second week of storage, i.e. during day 0and 4 of storage in the first week and during day 7 and 10 of storage inthe second week of the low pH treatment. For discussion, please refer toSection 4.1.1 (“Temperature profile”). Further samples were drawnaccording to the sampling plan (see Section 3.2), immediately diluted asdescribed above and titrated. After completion of the low pH storage at30° C.±1° C., the pH was determined by direct measurement and by themethod recommended by the European Pharmacopoeia (i.e., dilution to 1%protein with 0.9% NaCl solution).

Control Run without Virus

Two control runs were performed as described for the virus-spiked runs,where unspiked and 0.45 μm filtrated Subcuvia NG intermediate isprocessed. The same process parameters apply as specified for thevirus-spiked Runs 1 and 2, respectively, except that the material wasnot pH adjusted. Samples for determination of biochemical parameterswere taken according to the sampling plan (see Section 3.2).

The cytotoxicity of the mock-spiked starting material (spiked 0.9:10with BT-medium for infected cells *; sample is filtered as describedabove before titration) and the mock-spiked and pH adjusted Subcuvia NGintermediate after 0.45 μm filtration was determined. Samples fordetermination of cytotoxicity were taken according to the sampling plan(see Section 3.2).

-   -   * The composition of the BT-medium for infected cells is as        follows: DMEM (containing 4.5 g/l D-Glucose)+1% (v/v)        L-Glutamine (200 mM)+1% (v/v) Gentamicin Sulphate (10 mg/ml)+1%        (v/v) Sodium Pyruvate+2% (v/v) Sodium Bicarbonate (7.5%)+1%        (v/v) non-essential amino acids+5% (v/v) Horse serum.        Potential Interference

The interference of the sample matrix for samples containing Subcuvia NGintermediate after pH adjustment (pH 4.9±0.1) with the detection ofviruses was investigated in duplicate for each indicator cell line asfollows: a sample of 1 ml, drawn from the mock-spiked pH adjusted and0.45 μm filtered Subcuvia NG intermediate, was diluted 1:3.16 (v/v) withthe respective cold cell culture medium (stored at +2° C. to +8° C.).Subsequently, 1.8 ml of the diluted material was spiked 1:10 with 0.2 mlof pre-diluted* virus stock suspension to a calculated titer of 2.0 and3.0 log₁₀[TCID₅₀/ml]. After mixing, samples were drawn and titratedimmediately. As a control, 1.8 ml of the respective cold cell culturemedium (stored at +2° C. to +8° C.) was spiked the very same way withpre-diluted virus stock suspension before titration.

-   -   * The appropriate cell culture medium [BT-medium for BT cells        (BVDV), A9-medium for A9 cells (MMV)] was used for pre-dilution        of virus stock suspensions.        Sampling Plan        Virus Titrations

The following acceptance ranges apply for sample drawing: after 1 day ofstorage (±1 hour), after 2 to 6 days of storage (±2 hours), after 7 to13 days of storage (±3 hours), after 14 to 20 days of storage (±4hours), and after 21 to 27 days of storage (±5 hours). For Sample codes,see Section 1.1 (Process Scheme).

Samples were immediately titrated without additional storage.

Control stage Amount Virus stock suspension 1 × 0.3 ml   Virus-spikedstarting material 1 × 2 ml Virus-spiked and filtered starting material 1× 3 ml Virus-spiked, pH adjusted and filtered starting material^(§) 1 ×1 ml Virus-spiked, pH adjusted and filtered starting material stored atlow pH at 30 ± 1 × 1 ml 1° C., as soon as the temperature has reached29° C., i.e. “0 d” sample^(§), following each storage for 7 d^(§), 14d^(§), 20 d (only MMV)^(§) and 27 d (only MMV)^(§) Virus-spiked, pHadjusted and filtered starting material stored at low pH at 30 ± 1 × 6ml 1° C., following storage for 20 d and 27 d (only BVDV)^(§), titratedby TCID50 assay each as well as by bulk titration pH Hold control: pH7.0 ± 0.1, at 30 ± 1° C. 1 × 5 ml Temperature Hold control: pH 4.9 ±0.1§, at +2° C. to +8° C. 1 × 5 ml Combined pH and Temperature Holdcontrol: pH 7.0 ± 0.1, at +2° C. to +8° C. 1 × 5 ml ^(§)Samples wereimmediately diluted 1:3.16 with the respective cold cell culture mediumbefore titration.Determination of Cytotoxicity

Samples were immediately titrated without additional storage.

Amount (for each cell line and Control stage each run) Virus stocksuspension 1 × 0.3 ml   Mock-spiked and filtered starting material 1 × 3ml “SM Filt.” Mock-spiked pH adjusted and filtered 1 × 1 ml startingmaterial^(§) “SM pH Filt.” ^(§)Samples were immediately diluted 1:3.16with the respective cold cell culture medium before titration.Determination of Interference

Samples were immediately titrated without additional storage.

Control stage Amount Virus stock suspension 1 × 0.3 ml   Mock-spiked, pHadjusted (pH 4.9 ± 0.1) and filtered Subcuvia NG intermediate,, 1 × 1 mldiluted 1:3.16 with the respective cold cell culture medium, spiked 1:10with pre- diluted VSS to a calculated titer of 2.0 log₁₀[TCID₅₀/ml]Mock-spiked, pH adjusted (pH 4.9 ± 0.1) and filtered Subcuvia NGintermediate,, 1 × 1 ml diluted 1:3.16 with the respective cold cellculture medium, spiked 1:10 with pre- diluted VSS to a calculated titerof 3.0 log₁₀[TCID₅₀/ml] Cold cell culture medium spiked 1:10 withpre-diluted VSS to a calculated titer of 1 × 2 ml 2.0 log₁₀[TCID₅₀/ml]Cold cell culture medium spiked 1:10 with pre-diluted VSS to acalculated titer of 1 × 2 ml 3.0 log₁₀[TCID₅₀/ml] § Samples wereimmediately diluted 1:3.16 with the respective cold cell culture mediumbefore titration.Determination of Biochemical Parameters

Samples were drawn from each unspiked control run only and stored at +2to +8° C. until analysis.

Amount (incl. back-up Control stage Parameter samples) Starting materialbefore spiking¹ Molecular size distribution “SM-MSD” 2 × 1 ml CelluloseAcetate Electrophoresis “SM-CAE” Spiked starting material after lowMolecular size distribution “20 d-MSD” 2 × 1 ml pH storage at 30° C. ±1° C., for 20 Cellulose Acetate Electrophoresis “20 d-CAE” days² Spikedstarting material after low Molecular size distribution “27 d-MSD” 2 × 1ml pH storage at 30° C. ± 1° C., for 27 Cellulose AcetateElectrophoresis “27 d-CAE” days³ ¹Data generated during production of 2“Scale-Up”-lots as well as of the custom-made material (Lot. No IGSC64)were also taken into account for evaluation of the downscaled process.²Data generated during production of 2 “Scale-Up”-lots were also takeninto account for evaluation of the downscaled process. ³With regard tothe developmental stage of the product it seems unlikely that data ofbiochemical parameters after a 28 days storage period were available.Results and Discussion

The efficacy and robustness of the storage at low pH and elevatedtemperature of Subcuvia NG as a dedicated inactivation step for bothlipid- and non-lipid-enveloped viruses, was investigated with BVDV andMMV, using a down-scaled laboratory model of this step. Validationparameters and biochemical parameters were defined and measured in thedown-scaled process, and by comparison of the results with theconditions of the manufacturing process the equivalence of the twoprocesses was established.

Results for Validation and Biochemical Parameters

Validation Parameters

The validity of the down-scaled procedure was verified by determinationof the parameters critical for virus inactivation, i.e., the pH of theprocess material, the temperature during low pH storage and the time ofstorage.

pH Value Before Storage at Low pH and Elevated Temperature

The pH of the spiked process material was measured and adjusted to4.9±0.1 in all runs, as required and was therefore within the limitsspecified in the Study Plan. The pH values determined after storage arediscussed in Section 4.1.2 (Other Process and Biochemical Parameters).

Temperature Profile

To investigate the robustness of the storage step at low pH and elevatedtemperature against temperature variations, the temperature wasdecreased from 30° C.±1° C. to 25±1° C. for 6 hours once every week ofstorage. The temperature was always within the ranges specified in theStudy Plan. However, in the first and in the second week of storage, thedecrease in temperature to 25±1° C. for 6 hours was erroneouslyperformed twice per week, i.e., during day 0 and 4 in the first week andduring day 7 and 10 in the second week. As this incident shifted the runprofile to a worse scenario with respect to virus inactivation, therobustness of virus inactivation was even more extensively investigatedthan originally intended.

Storage Time

The duration of the storage at low pH and at elevated temperature is notspecified for the manufacturing process of Subcuvia NG. Depending on theoutcome of the current study, there are several options: one option forthe duration of the storage time is 21 to 22 days; a second option maybe approximately 28 days. To investigate the efficacy and robustness ofthe low pH treatment under conditions less favorable for virusinactivation all downscaled runs were performed below the lower limitsof the possible storage time; i.e., for 20 days±4 hours for the firstoption and storage for 27 days±5 hours for the second option. The spikedintermediate was stored at low pH and elevated temperature for 26days+22 hours (MMV, both runs) and for 27 days+1 hour (BVDV, both runs).In order to investigate virus clearance after 3 weeks of storage,samples for virus titration were drawn after 20 days+1 hour of storagefor MMV and after 20 days+2 hours for BVDV.

The storage time was within the specified limits for the down-scale,i.e., 20 days±4 hours, and 27 days±5 hours in all runs, which is belowthe currently considered lower limits of storage time in manufacturing.

Other Process and Biochemical Parameters

pH after Low pH Storage

Following low pH storage, the pH value was measured again directly aswell as according to the method recommended by the EuropeanPharmacopoeia (EP), i.e., dilution to 1% protein with 0.9% NaClsolution. The pH measured by direct measurement after the treatment wasclose to the pH value before low pH storage; i.e., the differencesranged from −0.07 to +0.04 (pH after minus pH before storage). The pHmeasured according to the EP method was always 0.2 to 0.3 highercompared to the direct measurement method. This slight increase of thepH values when using the EP method is typical for these two differentmethods in determination of the pH. This fact is also considered in thespecified limits at manufacturing scale, where the limits for the pHafter low pH treatment are 4.4 to 4.9 when determined by directmeasurement and 4.6 to 5.1 when determined by the EP method.

Molecular Size Distribution (MSD)

The molecular size distribution (by HPLC analysis) was investigatedbefore and after storage for the two unspiked control runs. The testresults were compared to three “Scale-Up” lots, produced during thedevelopmental stage of Subcuvia NG and to the custom made materialIGSC64 (14% as well as 20% protein concentration). Lot IGSC64, which isalso designated as a “Scale-Up” lot was not subjected to an storage atlow pH and elevated temperature in manufacture, as not enough materialwas available for this step. All results compared well to each other.Values for Molecular Size Distribution, especially percentage of IgGMonomers, were close to those observed in the “Scale Up” runs. For thesecond control run, where the concentration of IgG monomers afterstorage was a few percentage points lower, it is noted that already thematerial before storage had a comparatively low concentration of IgGmonomers, which also applies for the material tested within the“Scale-Up” process. Thus, the molecular size distribution data supportthe equivalence of the down-scale with the large-scale process.

Purity of Gammaglobulin

Purity of gammaglobulin (by CA-electrophoresis) of samples from the twounspiked control runs were determined and compared to the resultsobtained from two “Scale-Up” lots and from the custom made materialIGSC64 (14% as well as 20% protein concentration, values only before lowpH treatment, for explanation see above). All values determined for thepurity of gammaglobulin during the down-scaled process compared well tothe results obtained for the “Scale Up” lots and were within the assayaccuracy (the relative standard deviation is 1.5%). These resultsdemonstrate the comparability of the down scaled to the manufacturingprocess.

Results for Virus Titration

Cytotoxicity

Cytotoxicity of intermediates obtained before storage at low pH andelevated temperature was investigated in control runs using mock-spikedmaterial adjusted to the respective pH as described for the virus spikedruns. The mock-spiked process intermediates before and after pHadjustment showed only a very weak cytotoxic effect on the cells used inall two runs, except in run 2 on the BT cells, where the pH adjustedintermediate showed no cytotoxic effect from the 1.0 log₁₀ dilution andbeyond. No significant differences in cytotoxicity were noticed for thevirus-spiked runs.

Interference Testing

Results from interference testing show no significant interference ofthe sample matrix with the detection of low titers of BVDV and MMV:Differences in virus titers between the spiked cell culture mediumcontrols and the spiked Subcuvia NG intermediates were between −1.0log₁₀ to 0.1 log₁₀, which is within the accuracy of the virus titrationassay. Thus, titers obtained during the virus inactivation runs were notdistorted by interference effects and represent the actual titer of thesample.

Virus Inactivation

Two runs were performed with each of the viruses BVDV and MMV, i.e. run1 at 13.5% protein concentration, and run 2 at 20.9% proteinconcentration. Both runs were performed at pH 4.9±0.1 and at 30±1° C.,where the temperature was decreased to 25±1° C. for a minimum of 6 hoursonce per week. Comparison of the results of the two runs revealed nosignificant differences in virus inactivation kinetics between the tworuns performed at the upper and the lower limit of the possible proteinconcentration of Subcuvia NG.

Employing conditions least favorable for virus inactivation, i.e. thelower limits of storage time and temperature, as well as upper limits ofpH, significant inactivation of BVDV after 20 days±4 hours of storageand complete inactivation of all the virus that could be spiked into therespective Subcuvia NG intermediate after 27 days±5 hours of storage wasdemonstrated for BVDV, irrespective of the conditions investigated. Inboth runs residual infectivity from BVDV could be still be detected onday 20. However, all BVDV was inactivated to below the limit ofdetection by the end of the 27 day storage. The reduction factors forMMV demonstrate a substantial contribution of this process step to theviral safety of the product, also with regard to non-lipid envelopedviruses. Individual virus titers and reduction factors for the virusesused are discussed in more detail further below. In addition, graphicalillustrations of the virus inactivation kinetics are given for eachvirus following the respective results-table.

BVDV

BVDV was inactivated nearly to the limit of detection (run 1) or to thelimit of detection (run 2) by day 20 of storage, providing for reductionfactors of 5.3 log₁₀ and 5.5 log₁₀ in runs 1 and 2, respectively. After27 days of storage all BVDV spiked into the Subcuvia NG intermediate wasinactivated to below the limit of detection for both runs, withreduction factors of >6.6 log₁₀ and >6.5 log₁₀ in runs 1 and 2,respectively. No significant differences could be observed in theinactivation kinetics of the two runs. Both intermediates in run 1 (with13.5% protein concentration)] and run 2 (with 20.9% proteinconcentration) showed comparable inactivation kinetics.

The low pH treatment at elevated temperature demonstrated effectiveinactivation of BVDV after storage for 20 days±4 hours, with acalculated mean reduction factor of 5.4 log₁₀. After 27 days of storageat low pH and elevated temperature, complete inactivation of BVDV tobelow the limit of detection was achieved, where a mean reduction factorof >6.6 log₁₀ was calculated.

MMV

For MMV reduction factors of 2.9 log₁₀ and 3.1 log₁₀ were calculated forrun 1 and 2, respectively, after 20 days of storage at low pH andelevated temperature. The calculated mean reduction factor is 3.0 log₁₀.After 27 days of low pH treatment reduction factors of 3.4 log₁₀ and 3.7log₁₀ were obtained, where the calculated mean reduction factor is 3.6log₁₀. These reduction factors demonstrate a substantial contribution ofthis process step to the viral safety profile of the manufacturingprocess with regard to Parvoviruses, which are very resistant towardsphysicochemical inactivation. The virus inactivation kinetics in bothruns was biphasic, with faster inactivation during the first 7 days andsomewhat slower inactivation during the following three weeks.

Hold Controls

To investigate the mechanism of virus inactivation, i.e., whethermediated by pH or by temperature or by a combination of the two, threehold controls were kept under the respective conditions.

Titration of the “Combined Hold Control” (pH 7.0±0.1) that were kept at2° C. to 8° C. for 27 days±5 hours resulted for both virusesinvestigated in a virus titer comparable with the virus spiked startingmaterial, except for MMV in run 2, where a small loss in titer (1.6log₁₀) was observed.

Storage at 30±1° C. and pH 7.0±0.1 (“pH Hold Control”) showed that thelipid enveloped virus BVDV was very sensitive to storage at elevatedtemperature. BVDV was inactivated by 4.6 log₁₀ in both runs. Also, thenon-lipid-enveloped virus MMV was inactivated by 3.3 and 3.8 log₁₀, inrun 1 and run 2, respectively.

Titration of the “Temperature Hold Controls” (t HC) after a 27 days±5hours storage at low pH and at 2° C. to 8° C. resulted for both virusesinvestigated in a virus titer comparable with the virus spiked startingmaterial. These results demonstrate that BVDV as well as MMV wereresistant to storage at low pH in the range of 4.4 to 4.9 at lowtemperature.

Taken together, the results of the three Hold Controls suggest that:

-   -   Temperature would be the most significant factor for the        inactivation of BVDV, with some contribution of the low pH        (based on the fact that the control at 30±1° C. and neutral pH        was substantially, but not completely inactivated after 27        days).    -   For inactivation of MMV, temperature seemed to be the only        relevant factor as the virus titers of the low pH kinetic        samples after 27 days was virtually identical with the control        at neutral pH and 30° C.

SUMMARY AND CONCLUSION

In the course of the present study, a down-scaled model of the storageat low pH and elevated temperature in the manufacture of Subcuvia NG wasestablished and its equivalence to the manufacturing procedure wasdemonstrated by determination of several process parameters. Inaddition, a comparison of biochemical parameters of intermediates fromthe down-scaled model and the manufacturing process further supportedthe equivalence of both processes, indicating that the reduction factorsobtained during the down-scale are also valid for the large scale. Toinvestigate the robustness with regard to virus inactivation, the impactof different protein concentrations of Subcuvia NG was investigated, thepH of the spiked starting material was adjusted to the upper limitcompared to the manufacturing process, and the impact of periodicdecreases in temperature was investigated. The virus reduction factorsand the inactivation kinetics obtained in the current study demonstratethat the lipid-enveloped virus BVDV is effectively and robustlyinactivated by storage at low pH and elevated temperature within 27days. Also, after storage at low pH and elevated temperature for 20 daysa significant inactivation of BVDV with a mean calculated reductionfactor of 5.4 log₁₀ was achieved for BVDV. The calculated mean reductionfactors for the parvovirus model MMV, i.e., 3.0 log₁₀ after 20 days ofstorage and 3.6 log₁₀ after 20 days of storage, show that this processstep further contributes to the viral safety of the manufacturingprocess with regard to small non lipid-enveloped DNA viruses for highphysicochemical resistance [2], for an storage time of 20 as well as of27 days.

Both protein concentrations investigated showed the same inactivationkinetics for MMV and BVDV, suggesting that the impact of proteinconcentration on the virus inactivation capacity is not significant forconcentrated Immunoglobulin solutions and the viruses BVDV and MMV.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method for preparing a concentratedimmunoglobulin G (IgG) composition, comprising the steps: (A)concentrating a first solution comprising IgG to a protein concentrationof from 2% to 10% (w/v) by ultrafiltration using a firstultra-/diafiltration system comprising a first ultrafiltration membranehaving a nominal molecular weight cut off (NMWCO) of 100 kDa or less,thereby forming a first IgG concentrate, wherein at least 95% of proteinin the first solution is IgG; (B) diafiltering the first IgG concentrateagainst a diafiltration buffer using the first ultra-/diafiltrationsystem comprising the first ultrafiltration membrane, thereby forming afirst IgG diafiltrate; (C) concentrating the first IgG diafiltrate to aprotein concentration of greater than 20% (w/v) by ultrafiltration usingthe first ultra-/diafiltration system comprising the firstultrafiltration membrane, thereby forming a second IgG concentrate; (D)collecting the second IgG concentrate from the firstultra-/diafiltration system; (E) washing the first ultrafiltrationmembrane by re-circulating a post-wash buffer through the firstultra-/diafiltration system wherein the first ultra-/diafiltrationsystem is washed with a volume of post-wash buffer equal to at least twotimes the dead volume of the first ultra-/diafiltration system, therebyforming a first IgG post-wash solution; (F) transferring the first IgGpost-wash solution from the first ultra-/diafiltration system into asecond ultra-/diafiltration system comprising a second ultrafiltrationmembrane having a nominal molecular weight cut off (NMWCO) of 100 kDa orless, wherein the surface area of the second ultrafiltration membrane islower than the surface area of the first ultrafiltration membrane; (G)concentrating the first IgG post-wash solution to a proteinconcentration of greater than 20% (w/v) by ultrafiltration using thesecond ultra-/diafiltration system comprising a secondultra-/diafiltration membrane, thereby forming a third IgG concentrate;and (H) combining the third IgG concentrate from the secondultra-/diafiltration system with the second IgG concentrate, therebyforming a concentrated IgG composition.
 2. The method of claim 1,wherein the first and second ultrafiltration membranes have a nominalmolecular weight cut off (NMWCO) of 80 kDa or less.
 3. The method ofclaim 1, wherein the first and second ultrafiltration membranes have anominal molecular weight cut off (NMWCO) of 60 kDa or less.
 4. Themethod of claim 1, wherein the first and second ultrafiltrationmembranes have a nominal molecular weight cut off (NMWCO) of 50 kDa orless.
 5. The method of claim 1, wherein the first and secondultrafiltration membranes have a same nominal molecular weight cut off(NMWCO).
 6. The method of claim 2, wherein the first and secondultrafiltration membranes have a same nominal molecular weight cut off(NMWCO).
 7. The method of claim 3, wherein the first and secondultrafiltration membranes have a same nominal molecular weight cut off(NMWCO).
 8. The method of claim 4, wherein the first and secondultrafiltration membranes have a same nominal molecular weight cut off(NMWCO).
 9. The method of claim 1, wherein the surface area of thesecond ultrafiltration membrane is no more than a tenth of the surfacearea of the first ultrafiltration membrane.
 10. The method of claim 2,wherein the surface area of the second ultrafiltration membrane is nomore than a tenth of the surface area of the first ultrafiltrationmembrane.
 11. The method of claim 3, wherein the surface area of thesecond ultrafiltration membrane is no more than a tenth of the surfacearea of the first ultrafiltration membrane.
 12. The method of claim 4,wherein the surface area of the second ultrafiltration membrane is nomore than a tenth of the surface area of the first ultrafiltrationmembrane.
 13. The method of claim 1, further comprising the steps of:(I) washing the second ultrafiltration membrane by re-circulating apost-wash buffer through the second ultra-/diafiltration system, therebyforming a second IgG post-wash solution; and (J) combining the secondIgG post-wash solution from the second ultra-/diafiltration system withthe concentrated IgG composition formed in (H).
 14. The method of claim2, further comprising the steps of: (I) washing the secondultrafiltration membrane by re-circulating a post-wash buffer throughthe second ultra-/diafiltration system, thereby forming a second IgGpost-wash solution; and (J) combining the second IgG post-wash solutionfrom the second ultra-/diafiltration system with the concentrated IgGcomposition formed in (H).
 15. The method of claim 3, further comprisingthe steps of: (I) washing the second ultrafiltration membrane byre-circulating a post-wash buffer through the secondultra-/diafiltration system, thereby forming a second IgG post-washsolution; and (J) combining the second IgG post-wash solution from thesecond ultra-/diafiltration system with the concentrated IgG compositionformed in (H).
 16. The method of claim 4, further comprising the stepsof: (I) washing the second ultrafiltration membrane by re-circulating apost-wash buffer through the second ultra-/diafiltration system, therebyforming a second IgG post-wash solution; and (J) combining the secondIgG post-wash solution from the second ultra-/diafiltration system withthe concentrated IgG composition formed in (H).
 17. The method of claim5, further comprising the steps of: (I) washing the secondultrafiltration membrane by re-circulating a post-wash buffer throughthe second ultra-/diafiltration system, thereby forming a second IgGpost-wash solution; and (J) combining the second IgG post-wash solutionfrom the second ultra-/diafiltration system with the concentrated IgGcomposition formed in (H).
 18. The method of claim 6, further comprisingthe steps of: (I) washing the second ultrafiltration membrane byre-circulating a post-wash buffer through the secondultra-/diafiltration system, thereby forming a second IgG post-washsolution; and (J) combining the second IgG post-wash solution from thesecond ultra-/diafiltration system with the concentrated IgG compositionformed in (H).
 19. The method of claim 7, further comprising the stepsof: (I) washing the second ultrafiltration membrane by re-circulating apost-wash buffer through the second ultra-/diafiltration system, therebyforming a second IgG post-wash solution; and (J) combining the secondIgG post-wash solution from the second ultra-/diafiltration system withthe concentrated IgG composition formed in (H).
 20. The method of claim8, further comprising the steps of: (I) washing the secondultrafiltration membrane by re-circulating a post-wash buffer throughthe second ultra-/diafiltration system, thereby forming a second IgGpost-wash solution; and (J) combining the second IgG post-wash solutionfrom the second ultra-/diafiltration system with the concentrated IgGcomposition formed in (H).
 21. The method of claim 9, further comprisingthe steps of: (I) washing the second ultrafiltration membrane byre-circulating a post-wash buffer through the secondultra-/diafiltration system, thereby forming a second IgG post-washsolution; and (J) combining the second IgG post-wash solution from thesecond ultra-/diafiltration system with the concentrated IgG compositionformed in (H).
 22. The method of claim 10, further comprising the stepsof: (I) washing the second ultrafiltration membrane by re-circulating apost-wash buffer through the second ultra-/diafiltration system, therebyforming a second IgG post-wash solution; and (J) combining the secondIgG post-wash solution from the second ultra-/diafiltration system withthe concentrated IgG composition formed in (H).
 23. The method of claim11, further comprising the steps of: (I) washing the secondultrafiltration membrane by re-circulating a post-wash buffer throughthe second ultra-/diafiltration system, thereby forming a second IgGpost-wash solution; and (J) combining the second IgG post-wash solutionfrom the second ultra-/diafiltration system with the concentrated IgGcomposition formed in (H).
 24. The method of claim 12, furthercomprising the steps of: (I) washing the second ultrafiltration membraneby re-circulating a post-wash buffer through the secondultra-/diafiltration system, thereby forming a second IgG post-washsolution; and (J) combining the second IgG post-wash solution from thesecond ultra-/diafiltration system with the concentrated IgG compositionformed in (H).